Mycobacterium antigenic composition

ABSTRACT

Immunogenic compositions comprising an Rv1196 related antigen, wherein the conductivity of the composition is 13 mS/cm or lower, or the concentration of salts of the composition is 130 mM or lower, and their use in medicine, are provided.

FIELD OF THE INVENTION

The present invention relates to immunogenic compositions comprising anRv1196 related antigen and having a low ionic strength. The presentinvention also relates to such immunogenic compositions which furthercomprise one or more immunostimulants. Methods for the preparation ofsuch immunogenic compositions and related kits are also provided.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) is a chronic infectious disease caused by infectionwith Mycobacterium tuberculosis and other Mycobacterium species. It is amajor disease in developing countries, as well as an increasing problemin developed areas of the world. More than 2 billion people are believedto be infected with TB bacilli, with about 9.4 million new cases of TBand 1.7 million deaths each year. 10% of those infected with TB bacilliwill develop active TB, each person with active TB infecting an averageof 10 to 15 others per year. While annual incidence rates have peakedglobally, the number of deaths and cases is still rising due topopulation growth (World Health Organisation Tuberculosis Facts 2010).

The mycobacterial protein Rv1196 (described, for example, by the nameMtb39a in Dillon et al Infection and Immunity 1999 67(6): 2941-2950) orfragments or derivatives thereof are protein antigens of potentialbenefit for the treatment or prevention of tuberculosis. Rv1196 ishighly conserved, with 100% sequence identity across H37Rv, C, Haarlem,CDC1551, 94-M4241A, 98-R6041NH-RIF-EM, KZN605, KZN1435, KZN4207, KZNR506strains, the F11 strain having a single point mutation Q30K. Rv1196 is acomponent of the fusion protein antigens Mtb72f and M72 (described, forexample, in international patent application WO2006/117240).

The formulation of protein antigens is extremely important in order toensure immunogenicity is maintained. Immunostimulants are sometimes usedto improve the immune response raised to any given antigen. However, theinclusion of adjuvants into an immunogenic composition increases thecomplexity of preparation of the components as well as the complexity ofdistribution and formulation of the composition. The preparation of eachof the adjuvant components as well as the antigenic component must beconsidered by formulators. In particular, the compatibility of theantigenic component with the adjuvant component should be considered.This is particularly the case where lyophilised antigens or antigenicpreparations are intended to be reconstituted with an adjuvantpreparation. In such a circumstance, it is important that the buffer ofthe adjuvant preparation is suitable for the antigen and thatimmunogenicity or solubility of the antigen is not affected by theadjuvant.

SUMMARY OF THE INVENTION

The present inventors have identified for the first time that Rv1196related antigens are particularly sensitive to the presence of salts.Without being limited by theory, it is believed Rv1196 related antigensare detrimentally impacted by a phenomenon known as “salting out” whichmay be defined as the precipitation of a protein from its solution byinteraction with salts, such as sodium chloride. The present inventorshave found that these antigens aggregate and precipitate at aconcentration of sodium chloride as low as 150 mM. Consequently, thestability of immunogenic compositions comprising Rv1196 related antigenscan surprisingly be improved by a reduction in the concentration ofsodium chloride.

Accordingly, the present invention provides an immunogenic compositioncomprising an Rv1196 related antigen, wherein the conductivity of thecomposition is 13 mS/cm or lower.

Additionally provided is an immunogenic composition comprising an Rv1196related antigen, wherein the concentration of salts in said compositionis 130 mM or lower.

The present invention also provides an immunogenic compositioncomprising an Rv1196 related antigen, wherein the concentration ofsodium chloride in said composition is 130 mM or lower.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. QS21 lytic activity curve

FIG. 2. Percentage of each 3D-MPL congener in the different ASAformulations

FIG. 3. Nepholometry of immunogenic compositions with varied pH and NaClconcentrations after storage

FIG. 4. DLS of immunogenic compositions with varied pH and NaClconcentrations after storage

FIG. 5. DLS of immunogenic compositions with varied pH and NaClconcentrations after storage

FIG. 6. Nepholometry of immunogenic compositions with varied pH and NaClconcentrations after storage

FIG. 7. Antigenic stability of immunogenic compositions with varied pHand NaCl concentrations following after storage

FIGS. 8 a-8 d. SEC-HPLC analysis of immunogenic compositions with variedpH and NaCl concentrations after storage

FIG. 9. Antigenicity of immunogenic compositions with varied pH and NaClconcentrations after storage

FIG. 10. Conductivity of NaCl standard solutions

FIG. 11. Induction of CD4 T cell responses in mice using immunogeniccompositions of the invention

FIG. 12. Induction of CD8 T cell responses in mice using immunogeniccompositions of the invention

FIG. 13. Nepholometry of immunogenic compositions with varied pH andNaCl concentrations after storage

FIG. 14. DLS of immunogenic compositions with varied pH and NaClconcentrations after storage

FIG. 15. Antigenicity of immunogenic compositions with varied NaClconcentrations after storage

BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS

-   SEQ ID No: 1 Amino acid sequence for the Rv1196 protein from    Mycobacterium tuberculosis H37Rv-   SEQ ID No: 2 Nucleotide sequence encoding the Rv1196 protein from    Mycobacterium tuberculosis H37Rv-   SEQ ID No: 3 Amino acid sequence for the Rv1196 protein from    Mycobacterium tuberculosis F11-   SEQ ID No: 4 Nucleotide sequence encoding the Rv1196 protein from    Mycobacterium tuberculosis F11-   SEQ ID No: 5 Amino acid sequence for the M72 protein-   SEQ ID No: 6 Nucleotide sequence encoding the M72 protein-   SEQ ID No: 7 Amino acid sequence for the M72 protein with two    N-terminal His residues-   SEQ ID No: 8 Nucleotide sequence encoding the M72 protein with two    N-terminal His residues-   SEQ ID No: 9 Amino acid sequence for the Mtb72f protein-   SEQ ID No: 10 Nucleotide sequence encoding the Mtb72f protein-   SEQ ID No: 11 Amino acid sequence for the Mtb72f protein with six    N-terminal His residues-   SEQ ID No: 12 Nucleotide sequence encoding the Mtb72f protein with    six N-terminal His residues-   SEQ ID No: 13 Nucleotide sequence for CpG Oligo 1 (CpG 1826)-   SEQ ID No: 14 Nucleotide sequence for CpG Oligo 2 (CpG 1758)-   SEQ ID No: 15 Nucleotide sequence for CpG Oligo 3-   SEQ ID No: 16 Nucleotide sequence for CpG Oligo 4 (CpG 2006)-   SEQ ID No: 17 Nucleotide sequence for CpG Oligo 5 (CpG 1686)

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides an immunogeniccomposition comprising an Rv1196 related antigen, wherein theconductivity of the composition is 13 mS/cm or lower. In particular, thepresent invention provides immunogenic compositions comprising an Rv1196related antigen, wherein the conductivity of the immunogenic compositionis 12 mS/cm or lower, for example 10 mS/cm or lower, 8 mS/cm or lower, 6mS/cm or lower, 5 mS/cm or lower, 4 mS/cm or lower, or 3 mS/cm or lower.In a particular embodiment the conductivity of the immunogeniccomposition is 2.5 mS/cm or lower, such as 2.25 mS/cm or lower, or 2.0mS/cm or lower. In a further specific embodiment the conductivity of theimmunogenic composition is 1.5 to 2.5 mS/cm.

In a second aspect, the present invention provides an immunogeniccomposition comprising an Rv1196 related antigen, wherein theconcentration of salts in said composition is 130 mM or lower. Inparticular, the present invention provides immunogenic compositionscomprising an Rv1196 related antigen, wherein the concentration of saltsin said composition is 100 mM or lower, for example 90 mM or lower, 80mM or lower, 70 mM or lower, 60 mM or lower, 50 mM or lower, or 40 mM orlower. In a particular embodiment the concentration of salts in saidcomposition is 35 mM or lower, such as 30 mM or lower, or 25 mM orlower. In a further specific embodiment the concentration of salts insaid composition is 20 to 40 mM, such as 25 to 35 mM.

In a third aspect, the present invention provides an immunogeniccomposition comprising an Rv1196 related antigen, wherein theconcentration of sodium chloride is 130 mM or lower. In particular, thepresent invention provides immunogenic compositions comprising an Rv1196related antigen, wherein the concentration of sodium chloride is 100 mMor lower, for example 90 mM or lower, 80 mM or lower, 70 mM or lower, 60mM or lower, 50 mM or lower, 40 mM or lower, 30 mM or lower, 20 mM orlower or 15 mM or lower. In a particular embodiment the concentration ofsodium chloride in the immunogenic composition is 10 mM or lower, suchas 7.5 mM or lower. Suitably the concentration of sodium chloride in theimmunogenic composition or is at or below 5 mM. In a further specificembodiment, the immunogenic composition is essentially free of sodiumchloride. By essentially free is meant that the concentration of sodiumchloride is at or very near to zero mM (such as 3 mM or less, 2 mM orless or 1 mM or less).

Suitably, the concentration of CaCl₂ in the immunogenic compositionswill be 40 mM or lower, 30 mM or lower, 20 mM or lower, 15 mM or loweror 10 mM or lower.

Suitably, the concentration of MgSO₄ in the immunogenic compositionswill be 80 mM or lower, 60 mM or lower, 40 mM or lower, 30 mM or lower,20 mM or lower or 10 mM or lower.

Suitably, the total concentration of NH₄ ⁺, Mg²⁺ and Ca²⁺ ions in theimmunogenic compositions will be 80 mM or lower, 60 mM or lower, 40 mMor lower, 30 mM or lower, 20 mM or lower or 10 mM or lower.

The immunogenic compositions of the invention will be aqueouspreparations.

The conductivity of an immunogenic composition of the invention can bemeasured using techniques known in the art, for example using adedicated conductivity meter or other instrument with the capability tomeasure conductivity. One suitable instrument is the Zetasizer Nano ZSfrom Malvern Instruments (UK).

The skilled person can readily test for the concentration of both sodium(Na⁺) and chloride (CI) ions using known techniques and kits. Forexample, sodium can be determined using a kit such as the SodiumEnzymatic Assay Kit (Catalogue Number: BQ011EAEL) from Biosupply.Chloride can be determined using a kit such as Chloride Enzymatic AssayKit (Catalogue Number: BQ006EAEL) from Biosupply.

Tuberculosis (TB) is a chronic infectious disease caused by infectionwith Mycobacterium tuberculosis and other Mycobacterium species. It is amajor disease in developing countries, as well as an increasing problemin developed areas of the world. More than 2 billion people are believedto be infected with TB bacilli, with about 9.4 million new cases of TBand 1.7 million deaths each year. 10% of those infected with TB bacilliwill develop active TB, each person with active TB infecting an averageof 10 to 15 others per year. While annual incidence rates have peakedglobally, the number of deaths and cases is still rising due topopulation growth (World Health Organisation Tuberculosis Facts 2010).

Mycobacterium tuberculosis infects individuals through the respiratoryroute. Alveolar macrophages engulf the bacterium, but it is able tosurvive and proliferate by inhibiting phagosome fusion with acidiclysosomes. A complex immune response involving CD4+ and CD8+ T cellsensues, ultimately resulting in the formation of a granuloma. Central tothe success of Mycobacterium tuberculosis as a pathogen is the fact thatthe isolated, but not eradicated, bacterium may persist for longperiods, leaving an individual vulnerable to the later development ofactive TB.

Fewer than 5% of infected individuals develop active TB in the firstyears after infection. The granuloma can persist for decades and isbelieved to contain live Mycobacterium tuberculosis in a state ofdormancy, deprived of oxygen and nutrients. However, recently it hasbeen suggested that the majority of the bacteria in the dormancy stateare located in non-macrophage cell types spread throughout the body(Locht et al, Expert Opin. Biol. Ther. 2007 7(11):1665-1677). Thedevelopment of active TB occurs when the balance between the host'snatural immunity and the pathogen changes, for example as a result of animmunosuppressive event (Anderson P Trends in Microbiology 200715(1):7-13; Ehlers S Infection 2009 37(2):87-95).

A dynamic hypothesis describing the balance between latent TB and activeTB has also been proposed (Cardana P-J Inflammation & Allergy—DrugTargets 2006 6:27-39; Cardana P-J Infection 2009 37(2):80-86).

Although an infection may be asymptomatic for a considerable period oftime, the active disease is most commonly manifested as an acuteinflammation of the lungs, resulting in tiredness, weight loss, feverand a persistent cough. If untreated, serious complications and deathtypically result.

Tuberculosis can generally be controlled using extended antibiotictherapy, although such treatment is not sufficient to prevent the spreadof the disease. Actively infected individuals may be largelyasymptomatic, but contagious, for some time. In addition, althoughcompliance with the treatment regimen is critical, patient behaviour isdifficult to monitor. Some patients do not complete the course oftreatment, which can lead to ineffective treatment and the developmentof drug resistance.

Multidrug-resistant TB (MDR-TB) is a form which fails to respond tofirst line medications. 3.3% of all TB cases are MDR-TB, with anestimated 440,000 new MDR-TB cases occurring each year. Extensivelydrug-resistant TB (XDR-TB) occurs when resistance to second linemedications develops on top of resistance to first line medications. Thevirtually untreatable XDR-TB has been confirmed in 58 countries (WorldHealth Organisation Tuberculosis Facts 2010).

Even if a full course of antibiotic treatment is completed, infectionwith M. tuberculosis may not be eradicated from the infected individualand may remain as a latent infection that can be reactivated. In orderto control the spread of tuberculosis, an effective vaccinationprogramme and accurate early diagnosis of the disease are of utmostimportance.

Currently, vaccination with live bacteria is the most widely used methodfor inducing protective immunity. The most common Mycobacterium employedfor this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strainof M. bovis which was first developed over 60 years ago. However, thesafety and efficacy of BCG is a source of controversy—while protectingagainst severe disease manifestation in children, BCG does not preventthe establishment of latent TB or the reactivation of pulmonary diseasein adult life. Additionally, some countries, such as the United States,do not vaccinate the general public with this agent.

Several of the proteins which are strongly expressed during the earlystages of Mycobacterium infection have been shown to provide protectiveefficacy in animal vaccination models. However, vaccination withantigens which are highly expressed during the early stages of infectionmay not provide an optimal immune response for dealing with later stagesof infection. Adequate control during latent infection may require Tcells which are specific for the particular antigens which are expressedat that time. Post-exposure vaccines which directly target the dormantpersistent bacteria may aid in protecting against TB reactivation,thereby enhancing TB control, or even enabling clearance of theinfection. A vaccine targeting latent TB could therefore significantlyand economically reduce global TB infection rates.

Subunit vaccines based on late stage antigens could also be utilised incombination with early stage antigens to provide a multiphase vaccine.Alternatively, early and/or late stage antigens could be used tocomplement and improve BCG vaccination (either by boosting the BCGresponse or through the development of advanced recombinant BCGstrains).

The protein antigen Rv1196 is of potential benefit for the treatment orprevention of tuberculosis. Rv1196 is described, for example, by thename Mtb39a in Dillon et al Infection and Immunity 1999 67(6):2941-2950). The Rv1196 protein is highly conserved, with 100% sequenceidentity across H37Rv, C, Haarlem, CDC1551, KZN605, KZN1435 and KZN4207strains, the F11 strain having a single point mutation (Q30K).

The protein antigens Mtb72f and M72 are fusion proteins comprisingRv1196 and are of potential benefit for the treatment or prevention oftuberculosis. Mtb72f has been shown to provide protection in a number ofanimal models (see, for example: Brandt et al Infect. Immun. 200472(11):6622-6632; Skeiky et al J. Immunol. 2004 172:7618-7628; Tsenovaet al Infect. Immun. 2006 74(4):2392-2401; Reed et al PNAS 2009106(7):2301-2306). Mtb72f has also been the subject of clinicalinvestigations (Von Eschen et al 2009 Human Vaccines 5(7):475-482). M72is an improved antigen which incorporates a single serine to alaninemutation relative to Mtb72f, resulting in improved stabilitycharacteristics. M72 related antigens have also been shown to be ofvalue in a latent TB model (international patent applicationWO2006/117240).

As used herein the term ‘Rv1196 related antigen’ refers to the Rv1196protein provided in SEQ ID No: 1 or an immunogenic derivative thereof.As used herein the term “derivative” refers to an antigen that ismodified relative to the reference sequence. Immunogenic derivatives aresufficiently similar to the reference sequence to retain the immunogenicproperties of the reference sequence and remain capable of allowing animmune response to be raised against the reference sequence. Aderivative may, for example, comprise a modified version of thereference sequence or alternatively may consist of a modified version ofthe reference sequence.

The Rv1196 related antigen may for example contain fewer than 1000 aminoacid residues, such as fewer than 900 amino acid residues, in particularfewer than 800 amino acid residues.

T cell epitopes are short contiguous stretches of amino acids which arerecognised by T cells (e.g. CD4+ or CD8+ T cells). Identification of Tcell epitopes may be achieved through epitope mapping experiments whichare known to the person skilled in the art (see, for example, Paul,Fundamental Immunology, 3rd ed., 243-247 (1993); Beiβbarth et alBioinformatics 2005 21(Suppl. 1):i29-i37). In a diverse out-bredpopulation, such as humans, different HLA types mean that particularepitopes may not be recognised by all members of the population. As aresult of the crucial involvement of the T cell response intuberculosis, to maximise the level of recognition and scale of immuneresponse, an immunogenic derivative of Rv1196 is desirably one whichcontains the majority (or suitably all) T cell epitopes intact.

The skilled person will recognise that individual substitutions,deletions or additions to the Rv1196 protein which alters, adds ordeletes a single amino acid or a small percentage of amino acids is an“immunogenic derivative” where the alteration(s) results in thesubstitution of an amino acid with a functionally similar amino acid orthe substitution/deletion/addition of residues which do notsubstantially impact the immunogenic function.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. In general, such conservativesubstitutions will fall within one of the amino-acid groupings specifiedbelow, though in some circumstances other substitutions may be possiblewithout substantially affecting the immunogenic properties of theantigen. The following eight groups each contain amino acids that aretypically conservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M)    -   (see, e.g., Creighton, Proteins 1984).

Suitably such substitutions do not occur in the region of an epitope,and do not therefore have a significant impact on the immunogenicproperties of the antigen.

Immunogenic derivatives may also include those wherein additional aminoacids are inserted compared to the reference sequence. Suitably suchinsertions do not occur in the region of an epitope, and do nottherefore have a significant impact on the immunogenic properties of theantigen. One example of insertions includes a short stretch of histidineresidues (e.g. 2-6 residues) to aid expression and/or purification ofthe antigen in question.

Immunogenic derivatives include those wherein amino acids have beendeleted compared to the reference sequence. Suitably such deletions donot occur in the region of an epitope, and do not therefore have asignificant impact on the immunogenic properties of the antigen.

The skilled person will recognise that a particular immunogenicderivative may comprise substitutions, deletions and additions (or anycombination thereof).

The terms “identical” or percentage “identity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsub-sequences that are the same or have a specified percentage of aminoacid residues that are the same (i.e., 70% identity, optionally 75%,80%, 85%, 90%, 95%, 98% or 99% identity over a specified region), whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. This definition also refers to the compliment of a testsequence. Optionally, the identity exists over a region that is at least250 amino acids in length, such as 300 amino acids or 350 amino acids.Suitably, the comparison is performed over a window corresponding to theentire length of the reference sequence (as opposed to the derivativesequence).

For sequence comparison, one sequence acts as the reference sequence, towhich the test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are entered into a computer,subsequence coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. Default program parameterscan be used, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percentage sequence identitiesfor the test sequences relative to the reference sequence, based on theprogram parameters.

A “comparison window”, as used herein, refers to a segment in which asequence may be compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), bycomputerised implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Current Protocols in Molecular Biology(Ausubel et al., eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid coordinates for regions ofsequence comparison and by designating the program parameters. UsingPILEUP, a reference sequence is compared to other test sequences todetermine the percent sequence identity relationship using the followingparameters: default gap weight (3.00), default gap length weight (0.10),and weighted end gaps. PILEUP can be obtained from the GCG sequenceanalysis software package, e.g., version 7.0 (Devereaux et al., Nuc.Acids Res. 12:387-395 (1984)).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(website at www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPS) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as theneighbourhood word score threshold (Altschul et al., supra). Theseinitial neighbourhood word hits act as seeds for initiating searches tofind longer HSPS containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (N) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

In any event, immunogenic derivatives of a polypeptide sequence willhave essentially the same activity as the reference sequence. Byessentially the same activity is meant at least 50%, suitably at least75% and especially at least 90% activity of the reference sequence in anin vitro restimulation assay of PBMC or whole blood with specificantigens (e.g. restimulation for a period of between several hours to upto two weeks, such as up to one day, 1 day to 1 week or 1 to 2 weeks)that measures the activation of the cells via lymphoproliferation,production of cytokines in the supernatant of culture (measured byELISA, CBA etc) or characterisation of T and B cell responses by intraand extracellular staining (e.g. using antibodies specific to immunemarkers, such as CD3, CD4, CD8, IL2, TNF-alpha, IFN-gamma, CD40L, CD69etc) followed by analysis with a flowcytometer. Suitably, by essentiallythe same activity is meant at least 50%, suitably at least 75% andespecially at least 90% activity of the reference sequence in a T cellproliferation and/or IFN-gamma production assay.

Suitably the Rv1196 related antigen will comprise, such as consist of, asequence having at least 70% identity to SEQ ID No: 1, such as at least80%, in particular at least 90%, especially at least 95%, for example atleast 98%, such as at least 99%.

A specific example of an Rv1196 related antigen is Rv1196 fromMycobacterium tuberculosis strain H37Rv, as provided in SEQ ID No: 1.Consequently, in one embodiment of the invention the Rv1196 relatedantigen is a protein comprising SEQ ID No: 1. In a second embodiment ofthe invention the Rv1196 related antigen is a protein consisting of SEQID No: 1.

A further example of an Rv1196 related antigen is Rv1196 fromMycobacterium tuberculosis strain F11, as provided in SEQ ID No: 3. Inone embodiment of the invention the Rv1196 related antigen is a proteincomprising SEQ ID No: 3. In a second embodiment of the invention theRv1196 related antigen is a protein consisting of SEQ ID No: 3.

Typical Rv1196 related antigens will comprise, such as consist of, animmunogenic derivative of SEQ ID No: 1 having a small number ofdeletions insertions and/or substitutions. Examples are those havingdeletions of up to 5 residues at 0-5 locations, insertions of up to 5residues at 0-5 five locations and substitutions of up to 20 residues.

Other immunogenic derivatives of Rv1196 are those comprising, such asconsisting of, a fragment of SEQ ID No: 1 which is at least 250 aminoacids in length, such as at least 300 amino acids in length or at least350 amino acids in length.

Additional immunogenic derivatives of Rv1196 are those comprising, suchas consisting of, a fragment of SEQ ID No: 3 which is at least 250 aminoacids in length, such as at least 300 amino acids in length, at least350 amino acids in length or at least 380 amino acids in length.

Rv1196 related antigens may be prepared by methods previously described(e.g. Dillon et al Infection and Immunity 1999 67(6): 2941-2950;WO2006/117240), those provided in the Examples, or methods analogousthereto.

The immunogenic compositions may comprise one or more further antigeniccomponents. Such additional antigenic components need not themselves besensitive to the presence of salts in the composition.

Additional antigenic components may be intended to strengthen orcomplement the immune responses solicited by the Rv1196 related antigenin the field of tuberculosis prevention and therapy or additionalantigens could be associated with other pathogens and are intended foradministration with the Rv1196 related antigen for reasons ofconvenience. Where a number of antigenic components are present withinthe formulation, these may be provided in the form of individualpolypeptides or fusion proteins. In some circumstances additionalantigenic components may be provided as a polynucleotide (orpolynucleotides).

Particular immunogenic derivatives of the Rv1196 protein include fusionproteins comprising a sequence having at least 70% identity to SEQ IDNo: 1, such as at least 80%, in particular at least 90%, especially atleast 95%, for example at least 98%, such as at least 99%.

Exemplary fusion proteins containing an Rv1196 protein include: M72 (SEQID No: 5) and variants thereof such as those with additional Hisresidues at the N-terminus (e.g. two His residues, as provided in SEQ IDNo: 7; or a polyhistadine tag of five or particularly six His residues,which may be used for nickel affinity purification); Mtb72f (SEQ ID No:9) which contains the original serine residue that has been mutated inM72, as are Mtb72f proteins with additional His residues at theN-terminus (e.g. two H is residues; or a polyhistadine tag of five orparticularly six His residues, which may be used for nickel affinitypurification).

In certain embodiments the Rv1196 related antigen comprises, such asconsists of, a sequence having at least at least 90% identity to SEQ IDNo: 5, especially at least 95%, for example at least 98%, such as atleast 99%. In other embodiments the Rv1196 related antigen comprises,such as consists of, a sequence having at least at least 90% identity toSEQ ID No: 7, especially at least 95%, for example at least 98%, such asat least 99%.

It is well known that for parenteral administration solutions shouldhave a pharmaceutically acceptable osmolality to avoid cell distortionor lysis. A pharmaceutically acceptable osmolality will generally meanthat solutions will have an osmolality which is approximately isotonicor mildly hypertonic. Suitably the immunogenic compositions of thepresent invention will have an osmolality in the range of 250 to 750mOsm/kg, for example, the osmolality may be in the range of 250 to 550mOsm/kg, such as in the range of 280 to 500 mOsm/kg.

Osmolality may be measured according to techniques known in the art,such as by the use of a commercially available osmometer, for examplethe Advanced® Model 2020 available from Advanced Instruments Inc. (USA).

An “isotonicity agent” is a compound that is physiologically toleratedand imparts a suitable tonicity to a formulation to prevent the net flowof water across cell membranes that are in contact with the formulation.

Generally, sodium chloride (NaCl) is used as a tonicity agent. Thepresent inventors have shown for the first time that that Rv1196 relatedantigens are particularly sensitive to “salting out”, a process wherebythe proteins in solution aggregate or coagulate when in solutionscontaining high concentrations of salt. Consequently, alternative meansare provided for ensuring the immunogenic compositions of the inventionhave a pharmaceutically acceptable osmolality.

In a particular embodiment there are provided immunogenic compositionsfurther comprising a non-ionic tonicity agent. A non-ionic tonicityagent for use in an immunogenic composition will itself need to bepharmaceutically acceptable, e.g. suitable for use in humans, as well asbeing compatible with the Rv1196 related antigen and further compatiblewith other components such as the immunostimulant(s).

In one embodiment of the present invention, suitable non-ionic tonicityagents are polyols, sugars (in particular sucrose, fructose, dextrose orglucose) or amino acids such as glycine. In one embodiment the polyol isa sugar alcohol, especially a C3-6 sugar alcohol. Exemplary sugaralcohols include glycerol, erythritol, threitol, arabitol, xylitol,ribitol, sorbitol, mannitol, dulcitol and iditol. In a specific exampleof this embodiment, a suitable non-ionic tonicity agent is sorbitol. Theskilled person will recognise that an appropriate osmolality may beattained through the use of a mixture of different tonicity agents. In aparticular embodiment of the invention the non-ionic tonicity agent inthe compositions of the invention incorporates sucrose and/or sorbitol.

In one embodiment, a suitable concentration of polyol within theimmunogenic composition is between about 2.5 and about 15% (w/v), inparticular between about 2.5 and about 10% (w/v) for example betweenabout 3 and about 7% (w/v), such as between about 4 and about 6% (w/v).In a specific example of this embodiment, the polyol is sorbitol.

In another embodiment, the immunogenic composition comprises sucrose andsorbitol. In such circumstances the immunogenic composition may suitablycontain between about 2.5 and about 15% (w/v) of sucrose and betweenabout 2.5 and about 15% (w/v) of sorbitol, in particular between about2.5 and about 10% (w/v) of sucrose and between about 2.5 and about 10%(w/v) of sorbitol, for example, between about 3 and about 7% (w/v) ofsucrose and between about 3 and about 7% (w/v) of sorbitol, such asbetween about 4 and about 6% (w/v) of sucrose and between about 4 andabout 6% (w/v) of sorbitol.

The pH of the immunogenic compositions should be suitable for parenteraladministration. Typically the pH will be in the range of 6.0 to 9.0.Suitably the pH will be in the range 7.0 to 9.0, especially 7.25 to8.75, such as 7.5 to 8.5, in particular pH 7.75 to 8.25. A pH of about8.0 is of particular interest.

The pH may be controlled by the use of buffers, including for exampleTris or phosphate buffers.

In a particular embodiment of the invention, the immunogenic compositioncomprises one or more immunostimulants.

In one embodiment, the immunostimulant may be a saponin. A particularlysuitable saponin for use in the present invention is Quil A and itsderivatives. Quil A is a saponin preparation isolated from the SouthAmerican tree Quillaja saponaria Molina and was first described byDalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. für die gesamteVirusforschung, Vol. 44, Springer Verlag, Berlin, p243-254) to haveadjuvant activity. Purified fractions of Quil A have been isolated byHPLC which retain adjuvant activity without the toxicity associated withQuil A (WO88/09336), for example QS7 and QS21 (also known as QA7 andQA21). QS21 is a natural saponin derived from the bark of Quillajasaponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cellsand a predominant IgG2a antibody response. QS21 is a preferred saponinin the context of the present invention.

In a suitable form of the present invention, the saponin adjuvant withinthe immunogenic composition is a derivative of saponaria Molina Quil A,in particular an immunologically active fraction of Quil A, such as QS17or QS21, suitably QS21.

Desirably, QS21 is provided in a less reactogenic composition where itis quenched with an exogenous sterol, such as cholesterol for example.Several particular forms of less reactogenic compositions wherein QS21is quenched with cholesterol exist. In a specific embodiment, thesaponin/sterol is in the form of a liposome structure (such as describedin WO96/33739, Example 1). In this embodiment the liposomes suitablycontain a neutral lipid, for example phosphatidylcholine, which issuitably non-crystalline at room temperature, for example egg yolkphosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilaurylphosphatidylcholine. The liposomes may also contain a charged lipidwhich increases the stability of the lipsome-QS21 structure forliposomes composed of saturated lipids. In these cases the amount ofcharged lipid is suitably 1-20% w/w, such as 5-10%. The ratio of sterolto phospholipid is 1-50% (mol/mol), suitably 20-25%.

Suitable sterols include β-sitosterol, stigmasterol, ergosterol,ergocalciferol and cholesterol. In one particular embodiment, theimmunogenic composition comprises cholesterol as sterol. These sterolsare well known in the art, for example cholesterol is disclosed in theMerck Index, 11th Edn., page 341, as a naturally occurring sterol foundin animal fat.

Where the active saponin fraction is QS21, the ratio of QS21:sterol willtypically be in the order of 1:100 to 1:1 (w/w), suitably between 1:10to 1:1 (w/w), and especially 1:5 to 1:1 (w/w). Suitably excess sterol ispresent, the ratio of QS21:sterol being at least 1:2 (w/w). In oneembodiment, the ratio of QS21:sterol is 1:5 (w/w). The sterol issuitably cholesterol.

In another embodiment, the immunogenic composition comprises animmunostimulant which is a Toll-like receptor 4 (TLR4) agonist. By “TLRagonist” it is meant a component which is capable of causing a signalingresponse through a TLR signaling pathway, either as a direct ligand orindirectly through generation of endogenous or exogenous ligand (Sabroeet al, J Immunol 2003 p1630-5). A TLR4 agonist is capable of causing asignaling response through a TLR-4 signaling pathway. A suitable exampleof a TLR4 agonist is a lipopolysaccharide, suitably a non-toxicderivative of lipid A, particularly monophosphoryl lipid A or moreparticularly 3-de-O-acylated monophoshoryl lipid A (3D-MPL).

3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A.and is referred throughout the document as MPL or 3D-MPL see, forexample, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094.3D-MPL primarily promotes CD4+ T cell responses with an IFN-gamma (Th1)phenotype. 3D-MPL can be produced according to the methods disclosed inGB2220211A. Chemically it is a mixture of 3-de-O-acylated monophosphoryllipid A with 3, 4, 5 or 6 acylated chains. In the compositions of thepresent invention small particle 3D-MPL may be used to prepare theimmunogenic composition. Small particle 3D-MPL has a particle size suchthat it may be sterile-filtered through a 0.22 um filter. Suchpreparations are described in WO94/21292. Suitably, powdered 3D-MPL isused to prepare the immunogenic compositions of the present invention.

Other TLR4 agonists which can be used are alkyl glucosaminide phosphates(AGPs) such as those disclosed in WO98/50399 or U.S. Pat. No. 6,303,347(processes for preparation of AGPs are also disclosed), suitably RC527or RC529 or pharmaceutically acceptable salts of AGPs as disclosed inU.S. Pat. No. 6,764,840. Some AGPs are TLR4 agonists, and some are TLR4antagonists.

Other suitable TLR4 agonists are as described in WO2003/011223 and inWO2003/099195, such as compound I, compound II and compound IIIdisclosed on pages 4-5 of WO2003/011223 or on pages 3-4 of WO2003/099195and in particular those compounds disclosed in WO2003/011223 asER803022, ER803058, ER803732, ER804053, ER804057m ER804058, ER804059,ER804442, ER804680 and ER804764. For example, one suitable TLR-4 agonistis ER804057.

In a particular embodiment, the immunogenic composition comprises both asaponin and a TLR4 agonist. In a specific example, the immunogeniccomposition comprises QS21 and 3D-MPL.

A TLR-4 agonist, such as a lipopolysaccharide, such as 3D-MPL, can beused at amounts between 1 and 100 ug per human dose of the immunogeniccomposition. 3D-MPL may be used at a level of about 50 ug, for examplebetween 40 to 60 ug, suitably between 45 to 55 ug or between 49 and 51ug or 50 ug. In a further embodiment, the human dose of the immunogeniccomposition comprises 3D-MPL at a level of about 25 ug, for examplebetween 20 to 30 ug, suitable between 21 to 29 ug or between 22 to 28 ugor between 23 and 27 ug or between 24 and 26 ug, or 25 ug.

A saponin, such as QS21, can be used at amounts between 1 and 100 ug perhuman dose of the immunogenic composition. QS21 may be used at a levelof about 50 ug, for example between 40 to 60 ug, suitably between 45 to55 ug or between 49 and 51 ug or 50 ug. In a further embodiment, thehuman dose of the immunogenic composition comprises QS21 at a level ofabout 25 ug, for example between 20 to 30 ug, suitable between 21 to 29ug or between 22 to 28 ug or between 23 and 27 ug or between 24 and 26ug, or 25 ug.

Where both TLR4 agonist and saponin are present in the immunogeniccomposition, then the weight ratio of TLR4 agonist to saponin issuitably between 1:5 to 5:1, suitably between 1:2 to 2:1, such as about1:1. For example, where 3D-MPL is present at an amount of 50 ug or 25ug, then suitably QS21 may also be present at an amount of 50 ug or 25ug, respectively, per human dose of the immunogenic composition. Certainimmunogenic compositions of the present invention comprise QS21 and3D-MPL, at an amount of between 1 and 100 ug of each per human dose,such as at an amount of between 10 and 75 ug of each per human dose.Immunogenic compositions of the present invention may suitably compriseQS21 and 3D-MPL, at an amount of between 15 and 35 ug of each per humandose, such as at an amount of between 20 and 30 ug of each per humandose.

In one embodiment, the immunostimulant is a TLR9 agonist, for example asset out in WO2008/142133. In a specific example, said TLR9 agonist is animmunostimulatory oligonucleotide, in particular an oligonucleotidecontaining an unmethylated CpG motif. Such oligonucleotides are wellknown and are described, for example, in WO96/02555, WO99/33488 and U.S.Pat. No. 5,865,462. Suitable TLR9 agonists for use in the immunogeniccompositions described herein are CpG containing oligonucleotides,optionally containing two or more dinucleotide CpG motifs separated byat least three, suitably at least six or more nucleotides. A CpG motifis a cytosine nucleotide followed by a guanine nucleotide.

In one embodiment the internucleotide bond in the oligonucleotide isphosphorodithioate, or possibly a phosphorothioate bond, althoughphosphodiester and other internucleotide bonds could also be used,including oligonucleotides with mixed internucleotide linkages. Methodsfor producing phosphorothioate oligonucleotides or phosphorodithioateare described in U.S. Pat. No. 5,666,153, U.S. Pat. No. 5,278,302 andWO95/26204. Oligonucleotides comprising different internucleotidelinkages are contemplated, e.g. mixed phosphorothioate phophodiesters.Other internucleotide bonds which stabilise the oligonucleotide may beused.

Examples of CpG oligonucleotides suitable for inclusion in theimmunogenic compositions described herein have the following sequences.In one embodiment, these sequences contain phosphorothioate modifiedinternucleotide linkages.

OLIGO 1 (SEQ ID No: 9): TCC ATG ACG TTC CTG ACG TT (CpG 1826)OLIGO 2 (SEQ ID No: 10): TCT CCC AGC GTG CGC CAT (CpG 1758)OLIGO 3 (SEQ ID No: 11): ACC GAT GAC GTC GCC GGT GAC GGC ACC ACGOLIGO 4 (SEQ ID No: 12): TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006)OLIGO 5 (SEQ ID No: 13): TCC ATG ACG TTC CTG ATG CT (CpG 1668)

Alternative CpG oligonucleotides may comprise the sequences above inthat they have inconsequential deletions or additions thereto.

In one embodiment the immunostimulant is a tocol. Tocols are well knownin the art and are described in EP0382271. In a particular embodiment,the tocol is alpha-tocopherol or a derivative thereof such asalpha-tocopherol succinate (also known as vitamin E succinate).

The present invention also provides a process for making an immunogeniccomposition of the invention comprising the steps:

-   -   a. lyophilising an Rv1196 related antigen; and    -   b. reconstituting the lyophilised Rv1196 related antigen of        step a) with an aqueous solution wherein the conductivity of the        solution is 13 mS/cm or lower.

In certain embodiments the conductivity of the aqueous solution is 12mS/cm or lower, for example 10 mS/cm or lower, 8 mS/cm or lower, 6 mS/cmor lower, 5 mS/cm or lower, 4 mS/cm or lower, or 3 mS/cm or lower. In aparticular embodiment the conductivity of the aqueous solution is 2.5mS/cm or lower, such as 2.25 mS/cm or lower, or 2.0 mS/cm or lower.

Suitably, the conductivity of the aqueous solution is such that when thelyophilised antigen is reconstituted the resulting solution has aconductivity of 13 mS/cm or lower, such as 12 mS/cm or lower, forexample 10 mS/cm or lower, 8 mS/cm or lower, 6 mS/cm or lower, 5 mS/cmor lower, 4 mS/cm or lower, or 3 mS/cm or lower. In a particularembodiment the conductivity of the resulting solution is 2.5 mS/cm orlower, such as 2.25 mS/cm or lower, or 2.0 mS/cm or lower.

Further provided is a process for making an immunogenic composition ofthe invention comprising the steps:

-   -   a. lyophilising an RV1196 related antigen; and    -   b. reconstituting the lyophilised Rv1196 related antigen of        step a) with an aqueous solution wherein the concentration of        salts in said solution is 130 mM or lower.

In certain embodiments the concentration of salts in said aqueoussolution is 100 mM or lower, for example 90 mM or lower, 80 mM or lower,70 mM or lower, 60 mM or lower, 50 mM or lower, or 40 mM or lower. In aparticular embodiment the concentration of salts in said aqueoussolution is 35 mM or lower, such as 30 mM or lower, or 25 mM or lower.

Suitably, the concentration of salts in the aqueous solution is suchthat when the lyophilised antigen is reconstituted the resultingsolution has a concentration of salts of 130 mM or lower, such as 100 mMor lower, for example 90 mM or lower, 80 mM or lower, 70 mM or lower, 60mM or lower, 50 mM or lower, or 40 mM or lower. In a particularembodiment the concentration of salts in the resulting solution is 35 mMor lower, such as 30 mM or lower, or 25 mM or lower.

Additionally provided is a process for making an immunogenic compositionof the invention comprising the steps:

-   -   a. lyophilising an Rv1196 related antigen; and    -   b. reconstituting the lyophilised Rv1196 related antigen of        step a) with an aqueous solution wherein the concentration of        sodium chloride in said solution is 130 mM or lower.

In certain embodiments the concentration of sodium chloride in saidaqueous solution is 100 mM or lower, for example 90 mM or lower, 80 mMor lower, 70 mM or lower, 60 mM or lower, 50 mM or lower, or 40 mM orlower. In a particular embodiment the concentration of salts in saidaqueous solution is 35 mM or lower, such as 30 mM or lower, 20 mM orlower, or 15 mM or lower. Suitably the concentration of sodium chloridein the aqueous solution is at or below 5 mM.

Suitably, the concentration of sodium chloride in the aqueous solutionis such that when the lyophilised antigen is reconstituted the resultingsolution has a concentration of sodium chloride of 130 mM or lower, suchas 100 mM or lower, for example 90 mM or lower, 80 mM or lower, 70 mM orlower, 60 mM or lower, 50 mM or lower, or 40 mM or lower. In aparticular embodiment the concentration of sodium chloride in theresulting solution is 35 mM or lower, such as 30 mM or lower, or 25 mMor lower.

In one embodiment the aqueous solutions of step b) (above) comprise asaponin and/or a TLR4 agonist, for example QS21 and/or 3D-MPL. In afurther embodiment the saponin and/or TLR4 agonist are in a liposomalformulation. In one embodiment, the aqueous solutions comprise a TLR4agonist and a saponin in a liposomal formulation, and a non-ionictonicity agent as described herein, such as a polyol. In particular theaqueous solutions may comprise sorbitol.

Also provided is a kit comprising:

-   -   a. a lyophilised Rv1196 related antigen; and    -   b. an aqueous solution wherein the conductivity of the solution        is 13 mS/cm or lower.

In certain embodiments the conductivity of the aqueous solution is 12mS/cm or lower, for example 10 mS/cm or lower, 8 mS/cm or lower, 6 mS/cmor lower, 5 mS/cm or lower, 4 mS/cm or lower, or 3 mS/cm or lower. In aparticular embodiment the conductivity of the aqueous solution is 2.5mS/cm or lower, such as 2.25 mS/cm or lower, or 2.0 mS/cm or lower.

Suitably, the conductivity of the aqueous solution is such that when thelyophilised antigen is reconstituted the resulting solution has aconductivity of 13 mS/cm or lower, such as 12 mS/cm or lower, forexample 10 mS/cm or lower, 8 mS/cm or lower, 6 mS/cm or lower, 5 mS/cmor lower, 4 mS/cm or lower, or 3 mS/cm or lower. In a particularembodiment the conductivity of the resulting solution is 2.5 mS/cm orlower, such as 2.25 mS/cm or lower, or 2.0 mS/cm or lower.

Additionally provided is a kit comprising:

-   -   a. a lyophilised Rv1196 related antigen; and    -   b. an aqueous solution wherein the concentration of salts in        said solution is 130 mM or lower.

In certain embodiments the concentration of salts in said aqueoussolution is 100 mM or lower, for example 90 mM or lower, 80 mM or lower,70 mM or lower, 60 mM or lower, 50 mM or lower, or 40 mM or lower. In aparticular embodiment the concentration of salts in said aqueoussolution is 35 mM or lower, such as 30 mM or lower, or 25 mM or lower.

Suitably, the concentration of salts in the aqueous solution is suchthat when the lyophilised antigen is reconstituted the resultingsolution has a concentration of salts of 130 mM or lower, such as 100 mMor lower, for example 90 mM or lower, 80 mM or lower, 70 mM or lower, 60mM or lower, 50 mM or lower, or 40 mM or lower. In a particularembodiment the concentration of salts in the resulting solution is 35 mMor lower, such as 30 mM or lower, or 25 mM or lower.

Further, there is provided a kit comprising:

-   -   a. a lyophilised Rv1196 related antigen; and    -   b. an aqueous solution wherein the concentration of sodium        chloride in said solution is 130 mM or lower.

In certain embodiments the concentration of sodium chloride in saidaqueous solution is 100 mM or lower, for example 90 mM or lower, 80 mMor lower, 70 mM or lower, 60 mM or lower, 50 mM or lower, or 40 mM orlower. In a particular embodiment the concentration of salts in saidaqueous solution is 35 mM or lower, such as 30 mM or lower, 20 mM orlower, or 15 mM or lower. Suitably the concentration of sodium chloridein the solution is at or below 5 mM.

Suitably, the concentration of sodium chloride in the aqueous solutionis such that when the lyophilised antigen is reconstituted the resultingsolution has a concentration of sodium chloride of 130 mM or lower, suchas 100 mM or lower, for example 90 mM or lower, 80 mM or lower, 70 mM orlower, 60 mM or lower, 50 mM or lower, or 40 mM or lower. In aparticular embodiment the concentration of sodium chloride in theresulting solution is 35 mM or lower, such as 30 mM or lower, or 25 mMor lower.

Kits may be adapted to provide a single dose of the immunogeniccomposition, such as a single human dose, or multiple doses of theimmunogenic composition.

The aqueous solutions used in kits of the invention may be any of theaqueous solutions as defined herein. In a specific embodiment of theinvention, the aqueous solution comprises a TLR4 agonist and/or asaponin in the form of liposomes. In a particular embodiment, the TLR4agonist is 3D-MPL and the saponin is QS21. The aqueous solutions usedherein may comprise a tonicity agent, for example a polyol, such asorbitol.

In respect of the above mentioned kits and methods for the production ofimmunogenic compositions of the invention, it may be noted thatimmunostimulant(s) and tonicity agent(s) if present may be colyophilisedwith the antigen or contained with the aqueous solution as desired. Theaqueous solution may simply be water for injection and all othercomponents of the immunogenic composition are colyophilised with theantigen. Typically, at least some immunostimulant(s) and tonicityagent(s) are provided in the aqueous solution, which is particularlyappropriate if certain components are poorly compatible withlyophilisation such as liposomes. In one embodiment the aqueous solutioncomprises an immunostimulant. In a second embodiment the aqueoussolution comprises a tonicity agent, e.g. a non-ionic tonicity agent,such as a polyol, in particular sorbitol. In a third embodiment theaqueous solution comprises an immunostimulant and a tonicity agent, suchas a polyol, in particular sorbitol.

Kits may further comprise instructions directing the reconstitution ofthe lyophilised Rv1196 related antigen using the aqueous solution.

The immunogenic compositions according the invention may be used inmedicine, in particular for the prophylaxis, treatment or ameliorationof infection by mycobacteria, such as infection by Mycobacteriumtuberculosis. The immunogenic compositions will generally be providedfor administration to humans, though they may also be of value inveterinary medicine such as for administration to bovines.

There is provided the use of an immunogenic composition according theinvention in the manufacture of a medicament, in particular a medicamentfor the prophylaxis, treatment or amelioration of infection bymycobacteria, such as infection by Mycobacterium tuberculosis.

There is also provided a method for the prophylaxis, treatment oramelioration of infection by mycobacteria, such as infection byMycobacterium tuberculosis, comprising the administration of a safe andeffective amount of an immunogenic composition according to the presentinvention.

The immunogenic composition may be provided for the purpose of:

-   -   treating active tuberculosis;    -   prophylaxis of active tuberculosis, such as by administering to        a subject who is uninfected, or alternatively a subject who has        latent infection;    -   treating latent tuberculosis;    -   prophylaxis of latent tuberculosis, such as by administering to        a subject who is uninfected; or    -   preventing or delaying reactivation of tuberculosis, especially        the delay of TB reactivation, for example by a period of months,        years or even indefinitely.

The term “active infection” refers to an infection, e.g. infection by M.tuberculosis, with manifested disease symptoms and/or lesions, suitablywith manifested disease symptoms.

The terms “inactive infection”, “dormant infection” or “latentinfection” refer to an infection, e.g. infection by M. tuberculosis,without manifested disease symptoms and/or lesions, suitably withoutmanifested disease symptoms. A subject with latent infection willsuitably be one which tests positive for infection, e.g. by PPD or Tcell based assays, but which has not demonstrated the disease symptomsand/or lesions which are associated with an active infection.

The term “primary tuberculosis” refers to clinical illness, e.g.,manifestation of disease symptoms, directly following infection, e.g.infection by M. tuberculosis. See, Harrison's Principles of InternalMedicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds.,2005).

The terms “secondary tuberculosis” or “postprimary tuberculosis” referto the reactivation of a dormant, inactive or latent infection, e.g.infection by M. tuberculosis. See, Harrison's Principles of InternalMedicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds.,2005).

The term “tuberculosis reactivation” refers to the later manifestationof disease symptoms in an individual that tests positive for infection(e.g. in a tuberculin skin test, suitably in an in vitro T cell basedassay) test but does not have apparent disease symptoms. Suitably theindividual will not have been re-exposed to infection. The positivediagnostic test indicates that the individual is infected, however, theindividual may or may not have previously manifested active diseasesymptoms that had been treated sufficiently to bring the tuberculosisinto an inactive or latent state.

Suitability an immunogenic composition is administered to a subject whois uninfected or who has a latent infection by mycobacteria, such asinfection by Mycobacterium tuberculosis.

The volume of immunogenic composition administered may vary dependingupon a number of other factors, such as the specific delivery route,e.g. intramuscular, subcutaneous or intradermal. Typically, the volumeadministered in a single injection (the unit dose) for a human will bein the range of 50 ul to 1 ml, such as 100 ul to 750 ul, especially 400to 600 ul, for example about 500 ul.

The quantity of Rv1196 related antigen contained within a single dose isdependent upon clinical needs but a single human dose will typically bein the range of 1 to 100 ug, such as 5 to 50 ug, for example 5 to 20 ug.A single human dose may contain about 10 ug of Rv1196 related antigen.

Suitably, compositions of the invention will be stable, in which ismeant that during storage at 25° C. for a period of 24 hoursantigenicity as measured by the techniques described herein remains atleast 80% of the antigenicity before storage. Desirably, antigenicitywill remain at least 85%, such as at least 90% and in particular atleast 95% after storage at 25° C. for a period of 24 hours. Forcompositions of particular interest, at least 80% of the antigenicity ofthe composition, such as at least 85%, at least 90% and especially atleast 95% remains after storage at 30° C. for a period of 24 hours.

The present invention will now be further described by means of thefollowing non-limiting examples.

EXAMPLES Example 1 Preparation of Adjuvant Composition ASA (Sorbitol)

An adjuvant composition was prepared which comprised 3-de-O-acylatedmonophosphoryl lipid A and QS21 in a liposomal formulation usingsorbitol as a tonicity agent. This was prepared as follows:

A. Method of Preparation of Liposomes:

A mixture of lipid (DOPC), cholesterol and 3-de-O-acylatedmonophosphoryl lipid A in organic solvent was dried down under vacuum.An aqueous solution (phosphate buffered saline [100 mM NaCl, 50 mMPhosphate pH 6.1]) was then added and the vessel agitated until all thelipid was in suspension. This suspension was then prehomogenised withhigh shear mixer and then high pressure homogenised until the liposomesize was reduced to around 90 nm±10 nm measured by DLS. Liposomes werethen sterile filtered.

B. ASA Formulation: Step 1: Dilution of Concentrated Liposomes

Na₂/K Phosphate buffer 100 mM pH 6.1 when diluted 10 times was added towater for injection to reach a 10 mM phosphate buffer concentration inthe final formulation. A 30% (w/v) sorbitol solution in water forinjection (WFI) was then added to reach a concentration of 4.7% in thefinal formulation—this was stirred for 15 to 45 minutes at roomtemperature.

Concentrated liposomes (made of DOPC, cholesterol and 3D-MPL at 40mg/ml, 10 mg/ml and 2 mg/ml respectively) were then added to the mix toreach a concentration of 100 ug/ml of 3D-MPL in the final formulation.

The mixture was subsequently stirred for 15 to 45 minutes at roomtemperature.

Step 2: QS21 Addition

Using a peristaltic pump, QS21 bulk stock was added to the dilutedliposomes under magnetic stirring to reach a 100 ug/ml concentration inthe final formulation. The mix was stirred for 15 to 45 minutes.

Final ASA (sorbitol) formulation contained 2 mg DOPC, 500 ugcholesterol, 100 ug 3D-MPL/ml and 100 ug QS21/ml, 4.7% sorbitol and 5 mMsodium chloride and 10 mM phosphate.

Step 3: pH was Checked to be 6.1±0.1 Step 4: Sterile Filtration

Sterile filtration was performed using a polyethersulfone (PES) filterfrom PALL Corporation.

Step 5: Storage at +2° C. to +8° C.

The adjuvant composition obtained, which comprised 3-de-O-acylated MPLand QS21 in a liposomal formulation and containing sorbitol as atonicity agent (designated ASA (sorbitol)), was then stored at 4° C.

Example 2 Preparation of Adjuvant Composition ASA (150 mM NaCl)

An adjuvant composition was prepared which comprised 3-de-O-acylatedmonophosphoryl lipid A and QS21 in a liposomal formulation using sodiumchloride as a tonicity agent.

A. Method of Preparation of Liposomes:

A mixture of lipid (DOPC), cholesterol and 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) in organic solvent was dried down undervacuum. Phosphate buffered saline (100 mM NaCl, 50 mM Phosphate pH 6.1)was then added and the vessel agitated until all the lipid was insuspension. This suspension was then prehomogenised with high shearmixer and then high pressure homogenised until the liposomes size wasreduced to around 90 nm±10 nm measured by DLS. Liposomes were thensterile filtered on 0.22 um PES membrane.

B. ASA Formulation: Step 1: Dilution of Concentrated Liposomes

Na₂/K Phosphate buffer 100 mM pH 6.45 when diluted 10 times and NaCl 1.5M were added to water for injection to reach respectively 10 mMphosphate and NaCl 150 mM concentrations in the final formulation. Thismixture was stirred for 5 minutes at room temperature. Concentratedliposomes (made of DOPC, cholesterol and 3D-MPL at 40 mg/ml, 10 mg/mland 2 mg/ml respectively) were then added to the mix to reach aconcentration of 100 ug/ml of 3D-MPL in the final formulation. Themixture was subsequently stirred for 5 to 15 minutes at roomtemperature.

Step 2: QS21 Addition

QS21 bulk stock was added to the diluted liposomes under magneticstirring to reach a 100 ug/ml concentration in the final formulation.The mix was stirred at room temperature.

Step 3: ph was Checked so as to be 6.1±0.1. Step 4: Sterile Filtration

Sterile filtration was performed using a polyethersulfone (PES) filterfrom PALL Corporation.

Step 5: Storage at +2° C. to +8° C.

Final composition of ASA (150 mM NaCl) was 2 mg DOPC, 500 ugcholesterol, 100 ug 3-de-O-acylated MPL, 100 ug QS21 per 1 ml, with 10mM phosphate and 150 mM NaCl.

Example 3 QS21 Lytic Activity

QS21 is known to lyse red blood cells (RBC). The ASA (sorbitol) adjuvantcomposition prepared as in Example 1 was tested to ensure that QS21lytic activity was quenched in the same way as was seen with theequivalent adjuvant composition comprising 150 mM NaCl (ASA (150 mMNaCl)).

QS21 lytic activity was measured by a haemolysis assay using chicken RedBlood cells (RBC). RBC were centrifuged at 550 g at 4° C. Supernatantwas discarded. The pellet was carefully resuspended in PBS buffer toreach the initial volume and the same operation was repeated untilsupernatant was no longer red (generally 3 times). The pellet was storedat 4° C. for 3 to 4 days maximum if not used directly (and washed againthe day it is used) or was diluted around 10 times in buffer if used thesame day.

A QS21 dose range curve was prepared in ASA buffer (in salt or insorbitol buffer following the ASA sample tested) extemporaneously andthe adjuvant samples (containing a 50 ug or 90 ug equivalent of QS21meaning the equivalent of 500 ul or 900 ul ASA) were prepared. Finalvolume was adjusted to 900 ul in standards and samples with adequatebuffer (containing or not sorbitol as a function of the buffer of thesample tested). Due to its opalescence, ASA interferes with opticaldensity (OD). ASA “blanks” were thus prepared and their OD wassubtracted from the OD of ASA tested samples. Those blanks correspondedto the same ASA volume as the volume tested in samples, but adjusted to1 ml with buffer. No RBC were added to these blanks. Standards andsamples were then incubated with RBC (100 ul of diluted RBC added to 900ul of standards and samples) for 30 minutes at room temperature (RT).Samples were then centrifuged 5 minutes at 900 g. Optical density at 540nm was measured after centrifugation.

Determination of lytic activity was carried out by a limit test.

1. Limit of detection (LOD) was defined as the lowest concentration ofQS21 leading to an OD:

-   -   Higher than the base level (OD>0.1)    -   Around three times higher than OD's buffer (the “0 ug” QS21)    -   In the ascendant part of the curve    -   Determined for each test.

2. QS21 lytic activity was held to be positive in the adjuvant samplesif the OD for the adjuvant sample was greater than the OD_(LOD)).

Example QS21 curve ug QS21 OD QS21 quenched 0 0.029 NA 0.5 0.052 <LOD0.6 0.073 <LOD 0.7 0.091 <LOD 0.8 0.096 <LOD 0.9 0.12 >98.2% 1 0.195  >98% 1.1 0.212 >97.8% 1.2 0.348 >97.6% 1.3 0.479 >97.4% 1.40.612 >97.2% 1.5 0.669   >97% 2 1.139   >96% 2.5 1.294   >95% 3 1.391  >94% 5 1.416   >90% Adjuvant* 0.03 >98.2% *50 ug QS21 equivalenttested. 150 mM sodium chloride buffer.

The above data is shown graphically in FIG. 1.

The Limit of Detection in this assay is at 0.9 ug QS21, and OD of 0.12

The QS21 quenching in an adjuvant composition comprising 150 mM sodiumchloride was estimated to be more than 98.2% for the equivalent of 50 ugQS21 tested. In the case of an equivalent of 90 ug tested, conclusion ismore than 99%.

QS21 quenching was then compared with an equivalent adjuvant compositioncomprising sorbitol and only 5 mM sodium chloride. Data were generatedafter storage of the ASA at 4° C. or after accelerated stability (7 daysat 37° C.). For the ASA in sorbitol, the QS21 standard curve wasrealised in a sorbitol containing buffer.

Sample Timepoint LOD QS21 quenched Adjuvant composition (ASA) T0<1.4 >97.2% 150 mM NaCl 7 days 37° C. <0.9 >98.2% Adjuvant Composition(ASA) T0 <2 >97.8% sorbitol, 5 mM NaCl 7 days 37° C. <1   >96% 11 months4° C. <2  >97.8%* Equivalent of 50 ug QS21 tested except * equivalent of90 ug QS21 tested.

It was concluded that QS21 was adequately quenched in a low sodiumchloride buffer.

Example 4 MPL Congeners

Chemically, 3D-MPL is a mixture of 3-de-O-acylated monophosphoryl lipidA with mainly 4, 5 or 6 acylated chains. Each separate 3D-MPL moleculeis called a congener. It is important that the congener compositionremain constant, with no shift between the proportion of congeners. Itis also important that any buffer used enables the congener compositionto be the same as in the concentrated liposomes used to make theadjuvant composition.

As shown on FIG. 2, the congener composition was examined in 3D-MPLconcentrated liposomes (Conc. Liposomes LIP07-217, first column of FIG.2), an adjuvant composition comprising 3D-MPL liposomes and QS21 in a150 mM NaCl buffer (Adjuvant 150 mM NaCl, or ASA (150 mM NaCl), secondcolumn), and an adjuvant composition comprising 3D-MPL liposomes andQS21 in a sorbitol and 5 mM NaCl buffer (Adjuvant Sorbitol, or ASA(sorbitol), columns 3-7).

The congener composition was also examined in two lots of ASA (sorbitol)adjuvant at day 0 and 7 days after preparation and maintenance at 37° C.to ensure that there was no evolution over time (see final four columnsof FIG. 2).

Relative distribution of tetra-, penta- and hexa-acylated congeners ofMPL in concentrated liposomes or ASA (sorbitol) samples was determinedby IP-HPLC-Fluo detection (ARD). Both standards and samples werederivatised with dansylhydrazine, which introduces a Fluo-activechromophore on the dissacharide backbone. The derivatised samples wereanalysed on a C18 reverse phase column using tetrabutylammoniumhydroxide (TBAOH) as an ion pair reagent. Congeners containing the samenumbers of fatty acyl groups were eluted in distinct groups (tetraacyl,pentaacyl, and hexaacyl).

Distribution of congeners is deduced by comparing the peak area of eachgroup to the total peak area of all MPL congeners.

FIG. 2 shows the percentage of each congener. No significant differencein congener composition was found between adjuvant buffers, and thecongener composition was consistent over time in the sorbitol buffer.

Example 5 Preparation of Adjuvant Composition ASA (Sorbitol-2)

An adjuvant composition was prepared which comprised 3-de-O-acylatedmonophosphoryl lipid A and QS21, at a reduced level relative to Example1, in a liposomal formulation using sorbitol as a tonicity agent. Thiswas prepared as follows:

The adjuvant was prepared by 1:1 dilution of ASA (sorbitol), preparedaccording to Example 1, with a solution containing 10 mM phosphate, 5 mMNaCl, 4.7% sorbitol at pH 6.1.

Final ASA (sorbitol-2) formulation contained 1 mg DOPC, 250 ugcholesterol, 50 ug 3D-MPL/ml and 50 ug QS21/ml, 4.7% sorbitol, 5 mMsodium chloride and 10 mM phosphate.

Example 6 Preparation of Adjuvant Composition ASA (Sorbitol-3)

An adjuvant composition was prepared which comprised 3-de-O-acylatedmonophosphoryl lipid A and QS21, at a reduced level relative to Example1, in a liposomal formulation using sorbitol as a tonicity agent. Thiswas prepared as follows:

A. Method of Preparation of Liposomes:

A mixture of lipid (DOPC), cholesterol and 3-de-O-acylatedmonophosphoryl lipid A in organic solvent was dried down under vacuum.An aqueous solution (phosphate buffered saline [100 mM NaCl, 50 mMPhosphate pH 6.1]) was then added and the vessel agitated until all thelipid was in suspension. This suspension was then prehomogenised withhigh shear mixer and then high pressure homogenised until the liposomessize was reduced to around 90 nm±10 nm measured by DLS. Liposomes werethen sterile filtered.

B. ASA Formulation: Step 1: Dilution of Concentrated Liposomes

Na₂/K Phosphate buffer 100 mM pH 6.1 when diluted 10 times was added towater for injection to reach a 10 mM phosphate buffer concentration inthe final formulation. A 30% (w/v) sorbitol solution in water forinjection (VVFI) was then added to reach a concentration of 4.7% in thefinal formulation—this was stirred for 15 to 45 minutes at roomtemperature.

Concentrated liposomes (made of DOPC, cholesterol and 3D-MPL at 40mg/ml, 10 mg/ml and 2 mg/ml respectively) were then added to the mix toreach a concentration of 50 ug/ml of 3D-MPL in the final formulation.

The mixture was subsequently stirred for 15 to 45 minutes at roomtemperature.

Step 2: QS21 Addition

Using a peristaltic pump, QS21 bulk stock was added to the dilutedliposomes under magnetic stirring to reach a 50 ug/ml concentration inthe final formulation. The mix was stirred for 15 minutes.

Final ASA formulation contained 1 mg DOPC, 250 ug cholesterol, 50 ug3D-MPL/ml and 50 ug QS21/ml, 4.7% sorbitol and 2.5 mM sodium chloride,10 mM phosphate.

Step 3: pH was Checked to be 6.1±0.1 Step 4: Sterile Filtration

Sterile filtration was performed using a polyethersulfone (PES) filterfrom PALL Corporation.

Step 5: Storage at +2° C. to +8° C.

The adjuvant composition obtained, which comprised 3-de-O-acylated MPLand QS21 in a liposomal formulation and containing sorbitol as atonicity agent (designated ASA (sorbitol-3)), was then stored at 4° C.

Example 7 Preparation of Adjuvant Composition ASA (150 mM NaCl-2)

An adjuvant composition was prepared which comprised 3-de-O-acylatedmonophosphoryl lipid A and QS21, at a reduced level relative to Example2, in a liposomal formulation using sodium chloride as a tonicity agent.This was prepared as follows:

A. Method of Preparation of Liposomes:

A mixture of lipid (DOPC), cholesterol and 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) in organic solvent was dried down undervacuum. Phosphate buffered saline (100 mM NaCl, 50 mM Phosphate pH 6.1)was then added and the vessel agitated until all the lipid was insuspension. This suspension was then prehomogenised with high shearmixer and then high pressure homogenised until the liposomes size wasreduced to around 90 nm±10 nm measured by DLS. Liposomes were thensterile filtered on 0.22 um PES membrane.

B. ASA Formulation: Step 1: Dilution of Concentrated Liposomes

Na₂/K Phosphate buffer 100 mM pH 6.45 when diluted 10 times and NaCl 1.5M were added to water for injection to reach respectively 10 mMphosphate and NaCl 150 mM concentrations in the final formulation. Thismixture was stirred for 5 minutes at room temperature. Concentratedliposomes (made of DOPC, cholesterol and 3D-MPL at 40 mg/ml, 10 mg/mland 2 mg/ml respectively) were then added to the mix to reach aconcentration of 50 ug/ml of 3D-MPL in the final formulation. Themixture was subsequently stirred for 5 to 15 minutes at roomtemperature.

Step 2: QS21 Addition

QS21 bulk stock was added to the diluted liposomes under magneticstirring to reach a 50 ug/ml concentration in the final formulation. Themix was stirred at room temperature.

Step 3: ph was checked so as to be 6.1±0.1.

Step 4: Sterile Filtration

Sterile filtration was performed using a polyethersulfone (PES) filterfrom PALL Corporation.

Step 5: Storage at +2° C. to +8° C.

Final composition of ASA (150 mM NaCl-2) was 1 mg DOPC, 250 ugcholesterol, 50 ug 3-de-β-acylated MPL, 50 ug QS21 per 1 ml, 10 mMphosphate and 150 mM NaCl.

Example 8 Investigation of “Salting Out” in Compositions ComprisingRv1196 with Different Salt Concentrations at pH 6.1, 7.5 and 8.5

The impact of sodium chloride concentration and pH on Rv1196 antigenstability, as assessed by size, was investigated.

Method

Rv1196 protein (SEQ ID No: 1) was obtained from Corixa Corporation andwas diluted to a concentration of 100 ug/ml in three different buffers(10 mM phosphate buffer at pH 6.1, 20 mM Tris buffer at pH 7.5 and 20 mMTris buffer at pH 8.5) containing final sodium chloride concentrationsof 0, 150 and 450 mM.

Samples were stored for 24 hours at 4° C. or 25° C. before analysis.

Nephlometry was performed using a Nepheloskan® Ascent, available fromThermo Fischer Scientific. Analysis was performed in UV transparentCostar® micro-plates available from Corning Inc (USA).

DLS was performed using a Dynapro Plate Reader from Wyatt Instruments.The instrument was operated using a laser wavelength of 830 nm and powerof 50 mW. Scattered light was detected at 150° at a temperature of 22°C. The mean hydrodynamic diameter and polydispersity index (p1) arecalculated by the instrument software.

Results

The findings of this experiment are presented in FIGS. 3 and 4.

Nephlometry demonstrates a general trend of deceased clarity withincreasing salt concentration, indicating that Rv1196 is sensitive tosalt concentration.

For the majority of DLS samples, instrumentation was unable to determinea specific particle size (shown as NV in FIG. 4). Nevertheless, wherecomparable data was obtained (i.e. pH 8.5 with 0 or 150 nM NaCl), it isclear that the concentration of sodium chloride impacts the measuredparticle size in Rv1196 immunogenic compositions.

Example 9 Preparation of Protein Antigens

M72 with Two N-Terminal his Residues (SEQ ID No: 7)

Construction of the M72 Expression Vector

A plasmid coding for the amino acid sequence of Mtb72f with anadditional 6-His tag at the N-terminus was generated by the sequentiallinkage in tandem of the open reading frames (ORFS) encoding the Cterminal fragment of Mtb32a to the full length ORF of Mtb39a followed atthe C terminus with the N terminal portion of Mtb32a. This wasaccomplished by using sequence-specific oligonucleotides containingunique restriction sites (EcoRI and EcoRV) and devoid of the stop codonsat the C terminal ends (in the case of the C terminal fragment of Mtb32aand Mtb39a) for polymerase chain reaction (PCR) of genomic DNA from theM. tuberculosis strain H37Rv. Using this vector as template, a mutationof Ser706 to Ala was performed by site-directed mutagenesis. The properorientation of inserts as well as the mutation Ser706Ala was verified byDNA sequencing.

In order to obtain the vector coding for M72, which just has 2 Hisresidues at the N terminus, four H is were deleted making use of acommercial site-directed mutagenesis system. After sequenceverification, the M72 coding sequence was excised from the plasmid byenzymatic reaction, gel purified and ligated into a pET vector. Therecombinant plasmid was then sequence verified. This plasmid codes forM72 under the control of a T7 promoter. Expression of T7 RNA polymeraseis driven from a genomic integrant in the expression host and is inducedusing a lac operon-based system (lacl) and an IPTG chemical inductionsignal. The expression plasmid is provided with kanamycin resistance.

The plasmid coding for the M72 fusion protein under the control of a T7promoter was transformed into the HMS174 (DE3) strain of E. coli, usingan electroporation method. The coding sequence of the M72 insert and theflanking regions were sequenced on both strands and were found to beidentical to the sequence determined from the original plasmidconstruct.

Fermentation

A vial of pelleted working seed was thawed at room temperature. Apre-dilution was prepared by mixing the working seed with 4.9 ml ofpre-culture medium. 1 ml of the pre-dilution was used to inoculate theliquid pre-culture which consists of 400 ml of pre-culture mediumsupplemented with 50 mg/l kanamycin sulfate and 10 g/l glucose.

Pre-culture medium composition Ingredient Concentration KH₂PO₄ 14.83 g/lK₂HPO₄ 1.65 g/l (NH₄)₂SO₄ 5.82 g/l Yeast extract 6.21 g/l Glycerol 87%(w/w) 14.54 ml/l Metal and salt solution⁽¹⁾: 9.7 ml/l FeCl₃6H₂O 3.3 g/lMgSO₄7H₂O 58 g/l Micro element solution⁽²⁾: 116 ml/l ZnSO₄7H₂O 7.65 g/lMnSO₄H₂O 5.28 g/l CuSO₄5H₂O 1.1 g/l CoCl₂6H₂O 1.1 g/l H₃BO₃ 0.3 g/lNa₂MoO₄2H₂O 2.64 g/l HCl 4N 6.2 ml/l Biotine and CaCl₂ solution⁽²⁾: 0.97ml/l Biotine 0.05 g/l CaCl₂2H₂O 61.7 g/l pH of the medium is adjusted to6.5 with NaOH (25%) solution The medium is filtered through 0.22 um⁽¹⁾pH adjusted to 1.50 with HCl (37%) solution; the solution is filteredthrough 0.22 um ⁽²⁾The solution is filtered through 0.22 um

The pre-culture was incubated in a 2 litre shake flask at 30° C. underagitation (200 RPM) until the OD_(650nm) reached a value between 2 and 4(approximate incubation time: 16 hours). At that stage, a 72 litre(total volume) fermenter containing 45 litres of culture mediumsupplemented with 34 mg/l kanamycin sulfate was inoculated with 52 mlliquid pre-culture.

Culture medium composition Ingredient Concentration MgSO₄7H₂O 0.63 g/lFeCl₃6H₂O 0.056 g/l Micro element solution⁽¹⁾: 1.91 ml/l ZnSO₄7H₂O 7.65g/l MnSO₄H₂O 5.28 g/l CuSO₄5H₂O 1.1 g/l CoCl₂6H₂O 1.1 g/l H₃BO₃ 0.3 g/lNa₂MoO₄2H₂O 2.64 g/l HCl 4N 6.2 ml/l HCl 37% 0.40 mL/L Yeast extract 35g/L (NH₄)₂SO₄ 2.10 g/l KH₂PO₄ 18.70 g/l Sodium glutamate 2.5 g/lGlycerol 87% 0.276 ml/l Glucose 20 g/l Biotine solution⁽²⁾: 0.22 ml/lBiotine 1 g/l CaCl₂2H₂O 0.21 g/l The solution is filtered through 0.22um ⁽¹⁾The solution is filtered through 0.22 um ⁽²⁾pH adjusted to 11.0with NaOH (25%) solution; the solution is filtered through 0.22 um

During the growth phase, pH was maintained at 6.8±0.2 by periodicaddition of 25% (v/v) NH₄OH and 25% (v/v) H₃PO₄. After incubation for 16hours at 30° C., fed-batch was started with feed medium.

Feed medium composition Ingredient Concentration MgSO₄7H₂O 1.98 g/lFeCl₃6H₂O 0.178 g/l Micro element solution⁽¹⁾: 6.02 ml/l ZnSO₄7H₂O 7.65g/l MnSO₄H₂O 5.28 g/l CuSO₄5H₂O 1.1 g/l CoCl₂6H₂O 1.1 g/l H₃BO₃ 0.3 g/lNa₂MoO₄2H₂O 2.64 g/l HCl 4N 6.2 ml/l HCl 37% 1.24 ml/l Sodium glutamate5 g/l Yeast extract 40 g/l Glycerol 87% 590 ml/l Biotine solution⁽²⁾: 2ml/l Biotine 1 g/l CaCl₂2H₂O 0.66 g/l The solution is filtered through0.22 um ⁽¹⁾The solution is filtered through 0.22 um ⁽²⁾pH adjusted to11.0 with NaOH (25%) solution; the solution is filtered through 0.22 um

The temperature was maintained at 30° C. for a further 2 hours, thenraised to 37° C. until the end of fermentation. The air flow wasconstantly set to 75 l/min and the dissolved oxygen kept at 17%saturation by feedback control of the agitation and pressure. Smallquantities of antifoam solution were added on demand automatically. Bythe time the OD_(650nm) reached a value of 50 (±5), 1 mMIsopropyl-beta-D-thiogalactopyranoside (IPTG) was added in order toinduce the expression of M72. Fermentation ended after 5 hours from thetime point induction was started. The cell culture was cooled down to15° C. under slight agitation and centrifuged (at 4° C.) to obtain cellpellets which were thereafter stored at −20° C. in aliquots.

Isolation of Inclusion Bodies

The cell pellets collected from the harvest were thawed at roomtemperature and disrupted in lysis buffer (10 mM Tris, 50 mM NaCl, pH8.0) with a high pressure homogenizer. Thereafter the cell lysate wacentrifuged and the resulting cell pellets (or inclusion bodies, IBs)were washed with wash buffer containing urea, Tris and NaCl. The IBswere solubilised with solubilisation buffer containing 8 M urea andfiltered through a 0.2 um membrane. This filtered solution was firstpurified by anion exchange chromatography using a Q Sepharose Fast Flow(QSFF) column. The elution of M72 takes place with a 6 M urea, 20 mMbis-Tris propane, 90 mM NaCl, pH 7.0 solution.

M72 collected was further purified by Hydroxyapatite chromatography(HA), from which it is eluted with a 6 M urea, 20 mM bis-Tris propane,250 mM NaCl, pH 7.0 solution. The collected fraction was concentratedwith a 30 kDa membrane cassette and diafiltered against 20 mM Tris, pH7.5. M72 was then sterilised through a 0.22 um filter. The purified bulkwas then aliquoted and stored at −70° C.

Example 10 Investigation of “Salting Out” in Compositions Comprising M72with Different Salt Concentrations at pH 6.1, 7.5 and 8.5

The impact of sodium chloride concentration and pH on M72 antigenstability, as assessed by size and antigenicity, was investigated.

Method

Purified bulk antigen (M72 with two N-terminal His residues, SEQ ID No:7, as prepared in Example 9) was diluted to a concentration of 100 ug/mlin three different buffers (10 mM phosphate buffer at pH 6.1, mM Trisbuffer at pH 7.5 and 20 mM Tris buffer at pH 8.5) containing finalsodium chloride concentrations of 0, 50, 150, 300 and 450 mM.

Samples were analysed immediately (T0), stored overnight at 4° C. beforeanalysis (T0 O/N) or stored at 25° C. for 24 hours before analysis(T24h25° C.).

DLS was performed using a Malvern Zetasizer Nano ZS from MalvernInstruments (UK). The instrument was operated using a laser wavelengthof 633 nm and power of 4 mW. Scattered light was detected at 173° at atemperature of 22° C. The Z-average diameter (Zav) and polydispersityindex (p1) are calculated by the instrument software.

Nephlometry was performed using a Nepheloskan® Ascent, available fromThermo Fischer Scientific. Analysis was performed in UV transparentCostar® micro-plates available from Corning Inc (USA).

Antigenicity was quantified by a sandwich ELISA in which the antigen iscaptured by a M72-specific rabbit polyclonal antibody and subsequentlyrevealed by a M72(Mtb39)-specific mouse monoclonal antibody. Allmeasured values are presented relative to the expected antigenicitybased on the purified bulk protein used to prepare the testedformulations.

Results

The findings of this experiment are presented in FIGS. 5 to 7.

The results demonstrate for the first time that the stability ofsolutions containing an Rv1196 related antigen, specifically M72, issensitive to both pH and sodium chloride concentration. The impact ofsodium chloride on antigen size and antigenicity is all the more notableas the pH is lower.

Antigen size and antigenicity are not stable at pH 6.1 even in theabsence of sodium chloride. The addition of 50 mM sodium chloride at pH6.1 led to a size increase from 35 nm (0 mM sodium chloride at T0) up to58 nm (T0) or 79 nm after 24 hours at 25° C.

Antigen size and antigenicity are relatively stable over 24 hours at 25°C. at pH 7.5 or 8.5, particularly in the absence of sodium chloride orat a sodium chloride concentration of 50 mM. Nevertheless, increasingthe concentration of sodium chloride to 150 mM or greater results in aclear increase in antigen size and reduction in antigenicity.

Consequently, the benefits of the present invention extend to sequencescontaining Rv1196 related antigens and not just to the Rv1196 antigensequence itself.

Example 11 Prevention of “Salting Out” in Compositions Comprising M72,Immunostimulants and Using Sorbitol as a Tonicity Agent

In order to compare the stability of immunogenic compositions containing150 mM NaCl with compositions using sorbitol as a tonicity agent, anumber of samples were monitored using SEC-HPLC and ELISA.

Method

Three different lyophilisation cakes were prepared, such that whencombined with the appropriate adjuvant formulations from Examples 5 and7 the desired pH would be obtained:

(a) M72 with Two N-Terminal his Residues—Target pH 8.5 in ReconstitutedVaccine

-   -   15.75% (w/v) sucrose solution (prepared in water for injection)        was added to water for injection to reach a sucrose        concentration of 6.3%. 3% (w/v) Tween80 solution (prepared in        water for injection) was then added to reach a concentration of        0.025%. Tris-HCl buffer 1 M pH 8.8 was then added to reach a 50        mM Tris buffer concentration. The mixture was magnetically        stirred for 5 minutes at room temperature. Purified bulk antigen        (M72 with two N-terminal His residues, SEQ ID No: 7, as prepared        in Example 9) was then added to reach a protein concentration of        25 ug/ml. The mixture was magnetically stirred for 10 minutes at        room temperature. The pH was checked and found to be 8.8.    -   0.5 ml of the mixture obtained was filled in 3 ml glass vials        then freeze dried.

(b) M72 with Two N-Terminal his Residues—Target pH 8.0 in ReconstitutedVaccine

-   -   15.75% (w/v) sucrose solution (prepared in water for injection)        was added to water for injection to reach a sucrose        concentration of 6.3%. 3% (w/v) Tween80 solution (prepared in        water for injection) was then added to reach a concentration of        0.025%. Tris-HCl buffer 1 M pH 8.8 was then added to reach a 20        mM Tris buffer concentration. The mixture was magnetically        stirred for 5 minutes at room temperature. Purified bulk antigen        (M72 with two N-terminal His residues, SEQ ID No: 7, as prepared        in Example 9) was then added to reach a protein concentration of        25 ug/ml. The mixture was magnetically stirred for 10 minutes at        room temperature. The pH was checked and found to be 8.8.    -   0.5 ml of the mixture obtained was filled in 3 ml glass vials        then freeze dried.

(c) M72 with Two N-Terminal his Residues—Target pH 7.5 in ReconstitutedVaccine

-   -   15.75% (w/v) sucrose solution (prepared in water for injection)        was added to water for injection to reach a sucrose        concentration of 6.3%. 3% (w/v) Tween80 solution (prepared in        water for injection) was then added to reach a concentration of        0.025%. Tris-HCl buffer 1 M pH 8.8 was then added to reach a        12.5 mM Tris buffer concentration. The mixture was magnetically        stirred for 5 minutes at room temperature. Purified bulk antigen        (M72 with two N-terminal His residues, SEQ ID No: 7, as prepared        in Example 9) was then added to reach a protein concentration of        25 ug/ml. The mixture was magnetically stirred for 10 minutes at        room temperature. The pH was checked and found to be 8.8.    -   0.5 ml of the mixture obtained was filled in 3 ml glass vials        then freeze dried.

The lyophilisation cakes described above were reconstituted with 625 ulof the adjuvant solutions prepared in Examples 5 and 7. Uponreconstitution with adjuvant solution, the following immunogeniccompositions were obtained:

(i) M72 with Two N-Terminal his Residues—ASA (150 mM NaCl-2) pH 8.5

-   -   10 ug antigen (20 ug/ml)    -   5% w/v sucrose    -   40 mM Tris    -   0.02% w/v Tween80    -   500 ug DOPC    -   125 ug cholesterol    -   25 ug 3D-MPL    -   25 ug QS21    -   150 mM NaCl    -   10 mM phosphate    -   pH 8.5

(ii) M72 with Two N-Terminal his Residues—ASA (150 mM NaCl-2) pH 8.0

-   -   10 ug antigen (20 ug/ml)    -   5% w/v sucrose    -   16 mM Tris    -   0.02% w/v Tween80    -   500 ug DOPC    -   125 ug cholesterol    -   25 ug 3D-MPL    -   25 ug QS21    -   150 mM NaCl    -   10 mM phosphate    -   pH 8.0

(iii) M72 with Two N-Terminal his Residues—ASA (150 mM NaCl-2) pH 7.5

-   -   10 ug antigen (20 ug/ml)    -   5% w/v sucrose    -   12.5 mM Tris    -   0.02% w/v Tween80    -   500 ug DOPC    -   125 ug cholesterol    -   25 ug 3D-MPL    -   25 ug QS21    -   150 mM NaCl    -   10 mM phosphate    -   pH 7.5

(iv) M72 with Two N-Terminal his Residues—ASA (Sorbitol-2) pH 8.5

-   -   10 ug antigen (20 ug/ml)    -   5% w/v sucrose    -   40 mM Tris    -   0.02% w/v Tween80    -   500 ug DOPC    -   125 ug cholesterol    -   25 ug 3D-MPL    -   25 ug QS21    -   5 mM NaCl    -   4.7% w/v sorbitol    -   10 mM phosphate    -   pH 8.5

(v) M72 with Two N-Terminal his Residues—ASA (Sorbitol-2) pH 8.0

-   -   10 ug antigen (20 ug/ml)    -   5% w/v sucrose    -   16 mM Tris    -   0.02% w/v Tween80    -   500 ug DOPC    -   125 ug cholesterol    -   25 ug 3D-MPL    -   25 ug QS21    -   5 mM NaCl    -   4.7% w/v sorbitol    -   10 mM phosphate    -   pH 8.0

(vi) M72 with Two N-Terminal his Residues—ASA (Sorbitol-2) pH 7.5

-   -   10 ug antigen (20 ug/ml)    -   5% w/v sucrose    -   12.5 mM Tris    -   0.02% w/v Tween80    -   500 ug DOPC    -   125 ug cholesterol    -   25 ug 3D-MPL    -   25 ug QS21    -   5 mM NaCl    -   4.7% w/v sorbitol    -   10 mM phosphate    -   pH 7.5

Sample Analysis

The reconstituted immunogenic compositions described above werecharacterised after storage at 25° C. or 30° C. (T0, T6 h and T24 h).

SEC-HPLC analysis was performed by injection on a TOSOH TSK-Gel5000Pwxl(ID 7.8 mm×30 cm) equilibrated in 20 mM Tris buffer pH 8.5, detection byUV at 210 nm and flow rate 0.5 ml/min.

Antigenicity was quantified by a sandwich ELISA in which the antigen iscaptured by a M72-specific rabbit polyclonal antibody and subsequentlyrevealed by a M72(Mtb39)-specific mouse monoclonal antibody. Allmeasured values are presented relative to the expected antigenicitybased on the purified bulk protein used to prepare the testedformulations.

Results

The results are shown in FIGS. 8 a-8 d and 9.

SEC-HPLC profiles are stable after reconstitution in low saltcompositions using sorbitol as a tonicity agent at each pH (i.e. pH 7.5,8.0 and 8.5). This may be contrasted with the SEC-HPLC profiles forimmunogenic compositions containing 150 mM NaCl, which show clearchanges between the initial profile obtained and those following storageat 25° C. or 30° C. This evolution becomes more intense when the pH ofthe 150 mM NaCl composition is lowered.

The same conclusions can be drawn in terms of antigenicity, withrecoveries remaining largely stable after reconstitution in low saltcompositions using sorbitol as a tonicity agent at each pH (i.e. pH 7.5,8.0 and 8.5) up to 24 h at 30° C.

Example 12 Conductivity Determination for Immunogenic Compositions ofthe Invention

The conductivity of a range of immunogenic compositions according to thepresent invention was measured and compared to the conductivity ofcontrol sodium chloride solutions and with an immunogenic compositioncontaining a conventional quantity of sodium chloride.

Method

A range of standards having sodium chloride concentrations of 0, 75,100, 150, 250 and 300 mM were prepared from a stock solution of 1500 mMsodium chloride by dilution in water for injection.

Immunogenic compositions were prepared using M72 with two N-terminal Hisresidues according to the procedures provided in Example 9. Toinvestigate the contribution from the antigen itself and any residualmaterials in the purified bulk, placebo lyophilisation cakes were alsoprepared by excluding the antigen component.

Using a Malvern Zetasizer Nano and 1.5 ml of each sample in foldedcapillary cells, a voltage of 30 to 150 V (determined automatically bythe instrument) was applied and the conductivity determined.

Results

Conductivity of sodium chloride standard solutions Sodium chlorideconcentration Conductivity mM mS/cm 0 0.0 75 8.2 100 10.7 150 15.6 25023.9 300 30.0

A standard curve, based on this data, is provided in FIG. 10.

Conductivity of test solutions Sodium Equivalent chloride sodium con-Conduc- chloride centration tivity concentration Description mM mS/cm mMASA(sorbitol-2) 5 1.46 9 Placebo pH 8.0/ASA(sorbitol-2) 5 1.95 14 M72 pH8.0/ASA(sorbitol-2) 5 1.96 14 Placebo pH 8.5/ASA(sorbitol-2) 5 2.36 18M72 pH 8.5/ASA(sorbitol-2) 5 2.28 17 ASA(150 mM NaCl-2) 150 16 159Placebo pH 8.5/ASA(150 mM 150 14.8 147 NaCl-2) M72 pH 8.5/ASA(150 mM 15015.3 152 NaCl-2)

As can be seen from the data above, the conductivity of solutions whichutilise 150 mM NaCl is significantly greater than that of solutionswhich make minimal use of NaCl.

The impact of the antigen and any components in the purified bulk isminimal, as placebo preparations have comparable conductivity to theirRv1196 related antigen containing counterparts.

Example 13 Immunogenicity Testing of Immunogenic Compositions of theInvention

The aim of the this Example was to determine whether or not formulationchanges to reduce the quantity of salt in immunogenic compositions ofthe invention, with a view to improving protein stability, had an impacton in vivo immunogenicity.

Method

Four immunogenic compositions were evaluated:

-   -   1. M72 with two N-terminal His residues pH 8.5/ASA (150 mM        NaCl-2)    -   2. M72 with two N-terminal His residues pH 8.5/ASA (Sorbitol-2)    -   3. M72 with two N-terminal His residues pH 8/ASA (Sorbitol-2)    -   4. M72 with two N-terminal His residues pH 7.5/ASA (Sorbitol-2)

The immunogenicity of these antigen containing compositions wasevaluated in C57BL/6 mice. For each of the four compositions, 30 C57BL/6mice were injected 3 times intramuscularly, on days 0, 14 and 28 with 1ug of antigen in 50 ul of adjuvant solution (prepared by the procedureprovided in Example 11). The elicited M72 specific T cell responses(both CD4 & CD8) were measured 6 days post last immunisation (6dPlll).

For the determination of M72-specific cellular responses, peripheralblood lymphocytes from 30 mice/group were collected and pooled (sixpools of five mice/group). A red blood cells lysis was performed beforeplating the cells in vitro. The cells were restimulated in vitro with apool of overlapping peptides (15-mer peptides with an 11 amino acidoverlap, at 1 ug/ml/peptide) covering the M72 sequence (without theN-terminal His residues). Cells remaining in medium (no peptidestimulation) were used to determine the background responses. Two hoursafter the co-culture with the peptide pool, brefeldin A was added to thewells (to inhibit cytokine excretion) and the cells were storedovernight at 4° C. The cells were subsequently stained for the followingmarkers: CD4, CD8, IL-2, IFN-gamma and TNF-alpha.

Results

Each datapoint in FIGS. 11 and 12 represents the background subtractedM72-specific CD4 or CD8 T cell response, respectively, of a pool ofperipheral blood lymphocytes from five mice six days after the thirdimmunisation. The response is expressed as the percentage of CD4 T cellsproducing IFN-gamma and/or IL-2 and/or TNF-alpha in response tosimulation with the M72 peptide pool. The bar represents the median ofthe responses for each group.

The results in FIGS. 11 and 12 show that comparable CD4 and CD8 T cellresponses are induced following three immunisations with each of thetest formulations. Consequently, it may be concluded that a reduction inthe quantity of salts present in the immunogenic compositions of thepresent invention does not lead to a compromise in the induced T cellresponses.

Example 14 Investigation of “Salting Out” in Compositions Comprising M72with CaCl₂ or MgSO₄ at pH 6.1 and 8.0

To investigate impact of other salts on M72 antigen stability, solutionswere prepared with a range of concentrations of CaCl₂ or MgSO₄ and atdifferent pH levels. Visual inspection was used as a readout ofstability.

Method

Purified bulk antigen (M72 with two N-terminal His residues, SEQ ID No:7) was diluted to a concentration of 100 ug/ml in two different buffers(10 mM succinate buffer at pH 6.1 and 10 mM Tris buffer at pH 8.0)containing specified quantities of salts (0 mM; 150 mM or 300 mM NaCl;40 mM, 80 mM or 160 mM CaCl₂; 87.5 mM, 175 mM or 430 mM MgSO₄).

Samples were analysed directly after preparation.

Using a Mettler Toledo conductivity meter and 6 ml of each sample in anunsiliconised glass vial, the conductivity was determined.

Results

Conductivity pH (mS/cm) pH Visual Group Salt Buffer (theoretical)(measured) (measured) Observation A   0 mM Succinate 6.1 1.1 6.3 Clear10 mM B NaCl Succinate 6.1 13.4 6.1 Clear  150 mM 10 mM C NaCl Succinate6.1 20.0 6.1 Clear  300 mM 10 mM D CaCl₂ Succinate 6.1 8.0 6.1Opalescent   40 mM 10 mM E CaCl₂ Succinate 6.1 11.2 5.8 Opalescent +large   80 mM 10 mM particles F CaCl₂ Succinate 6.1 20.2 5.8Opalescent + large  160 mM 10 mM particles G MgSO₄ Succinate 6.1 7.7 6.1Opalescent 87.5 mM 10 mM H MgSO₄ Succinate 6.1 12.4 5.9 Opalescent +very  175 mM 10 mM large particles I MgSO₄ Succinate 6.1 20.4 5.9Opalescent + very  430 mM 10 mM large particles J   0 mM Tris 8.0 0.4638.0 Clear 10 mM K NaCl Tris 8.0 12.13 8.0 Clear  150 mM 10 mM L NaClTris 8.0 21.1 8.0 Clear  300 mM 10 mM M CaCl₂ Tris 8.0 6.7 8.1 Largeparticles   40 mM 10 mM N CaCl₂ Tris 8.0 10.8 8.0 Opalescent + large  80 mM 10 mM particles O CaCl₂ Tris 8.0 19.7 8.0 Opalescent + large 160 mM 10 mM particles P MgSO₄ Tris 8.0 7.5 8.0 Large particles 87.5 mM10 mM Q MgSO₄ Tris 8.0 10.9 8.2 Opalescent + very  175 mM 10 mM largeparticles R MgSO₄ Tris 8.0 21.7 8.1 Opalescent + very  430 mM 10 mMlarge particles

The results demonstrate that solutions containing Rv1196 relatedantigens can be sensitive to salts other than sodium chloride. Theimpact of CaCl₂ or MgSO₄ appears to be more pronounced than for sodiumchloride at comparable concentrations or conductivity.

Example 15 Investigation of “Salting Out” in Compositions ComprisingMtb72f with Different Salt Concentrations at pH 6.1, 7.5 and 8.5

The impact of sodium chloride concentration and pH on Mtb72f antigenstability, as assessed by size, was investigated.

Method

Purified bulk antigen (Mtb72f with 6 his residues, SEQ ID No: 11) wasdiluted to a concentration of 100 ug/ml in three different buffers (10mM phosphate buffer at pH 6.1, 20 mM Tris buffer at pH 7.5 and 20 mMTris buffer at pH 8.5) containing final sodium chloride concentrationsof 0, 150 and 450 mM.

Samples were stored for 24 hours at 4° C. or 25° C. before analysis.

Nephelometry was performed using a Nepheloskan® Ascent, available fromThermo Fischer Scientific. Analysis was performed in UV transparentCostar® micro-plates available from Corning Inc (USA).

DLS was performed using a Dynapro Plate Reader from Wyatt Instruments.The instrument was operated using a laser wavelength of 830 nm and powerof 50 mW. Scattered light was detected at 150° at a temperature of 22°C. The mean hydrodynamic diameter and polydispersity index (p1) arecalculated by the instrument software.

Results

The findings of this experiment are presented in FIGS. 13 and 14.

Both DLS and nephlometry demonstrate a general trend that Mtb72f issensitive to salt concentration and pH, in a similar manner to M72 asshown in previous examples. Consequently, the benefits of the presentinvention apply to Rv1196 related antigens and not just to the Rv1196sequence itself.

In the case of a number of DLS samples, instrumentation was unable todetermine a specific particle size (shown as NV in FIG. 14).

Example 16 Prevention of “Salting Out” in Compositions Comprising M72,Immunostimulants and Using Sorbitol as a Tonicity Agent

In order to compare the stability of immunogenic compositions containing150 mM NaCl with compositions using sorbitol as a tonicity agent,samples were monitored using an alternative ELISA.

Method

Lyophilisation cake was prepared as described in Example 11(specifically method (a) such that when combined with the appropriateadjuvant formulations from Example 7a pH of 8.5 would be obtained.

The lyophilisation cake describe above were reconstituted with 625 ul ofthe adjuvant solutions prepared in Example 7. Upon reconstitution withadjuvant solution, the following immunogenic compositions were obtained:

(i) M72 with Two N-Terminal his Residues—ASA (150 mM NaCl-2) pH 8.5

-   -   10 ug antigen (20 ug/ml)    -   5% w/v sucrose    -   40 mM Tris    -   0.02% w/v Tween80    -   500 ug DOPC    -   125 ug cholesterol    -   25 ug 3D-MPL    -   25 ug QS21    -   150 mM NaCl    -   10 mM phosphate    -   pH 8.5

(Ii) M72 with Two N-Terminal his Residues—ASA (Sorbitol-2) pH 8.5

-   -   10 ug antigen (20 ug/ml)    -   5% w/v sucrose    -   40 mM Tris    -   0.02% w/v Tween80    -   500 ug DOPC    -   125 ug cholesterol    -   25 ug 3D-MPL    -   25 ug QS21    -   5 mM NaCl    -   4.7% w/v sorbitol    -   10 mM phosphate    -   pH 8.5

The reconstituted immunogenic compositions described above werecharacterised after storage at 30° C. (T24 h) and compared with anextemporaneously prepared sample (T0).

Antigenicity was quantified by an indirect sandwich ELISA in which theantigen is captured by a M72-specific rabbit polyclonal antibody andsubsequently revealed by a M72(Mtb39)-specific mouse monoclonalantibody. Briefly, the plate is coated with anti-M72 rabbit polyclonalantibody at the dilution of 1/8000 in Dulbecco's Phosphate BufferedSaline overnight at 4° C. and after four washes the plates were blockedfor 1 h at 37° C. with saturation buffer (PBS, 0.1% Tween 20, 1% BSA).After the washing step, protein standard (M72 purified bulk: 1950ug/ml), internal control (M72: 1768 ug/ml) and samples are loaded inwells from the first column of the plate at a concentration ofapproximately 0.25 pg/ml and then a 2-fold serial dilution is performedin the saturation buffer (PBS, 0.025% Tween 20) from well 1 to 12 andincubated 1h30 at 37° C. After the washing step, the immune complex isthen incubated 1 h at 37° C. with anti-M72 mouse monoclonal antibody ata dilution of 1/1000 in saturation buffer (PBS, 0.025% Tween 20). Afterfour washes, a biotinylated rabbit anti-mouse polyclonal antibody wasadded at a dilution of 1/1000 in saturation buffer (PBS, 0.025% Tween20). After four washes, the signal was amplified by addingStreptavidin-Horseradish Peroxidase diluted 1/4000 in saturation buffer(PBS, 0.025% Tween 20). After four washes, the signal was revealed byortho phenylene diamine dihydrochlorid (OPDA) for 15 min at RT and thereaction is stopped by addition of HCl 1M. The coloration isproportional to the quantity of bound anti-M72 antibody, and is measuredat 490 nm and 620 nm. All washing steps were performed using PBS, 0.025%Tween 20.

All measured values are presented relative to the expected antigenicitybased on the purified bulk protein used to prepare the testedformulations.

Results

The results are shown in FIG. 15. Diamonds indicating the specificmeasurements for each of the three test samples, with a line indicatingthe average value.

Antigen recovery is largely stable after reconstitution in low saltcompositions using sorbitol as a tonicity agent at pH 8.5 up to 24 h at30° C. Recovery in ASA (sorbitol-2) was 83.5% after 24 hours (T0 87.1%,meaning 95.9% of the relative antigenicity was maintained), whereasrecovery in ASA (NaCl-2) was 54.5% after 24 hours (T0 81.0%, meaningonly 67.3% of the relative antigenicity was maintained after storage).

In summary, Example 8 demonstrates for the first time the detrimentalimpact resulting from pH and NaCl concentration on the stability ofimmunogenic compositions containing an Rv1196 related antigen. Example14 extends this work to show that other salts may also have adetrimental impact on the stability of immunogenic compositionscontaining an Rv1196 related antigen, with Examples 10, 11, 12, 15 and16 demonstrating that the effect is also applicable to other sequences.

Reformulation of the immunogenic compositions with a non-ionic tonicityagent addresses the antigen stability problems. Additionally, Examples3, 4, 13 and 16 demonstrate the removal of substantially all NaCl fromthe immunogenic formulation and its replacement with sorbitol as atonicity agent does not have a detrimental impact on the stimulation ofT cell responses.

Stability of immunogenic compositions is key and may be particularlychallenging when in isolated locations were refrigeration may not bereadily accessible. By reducing the presence of salts in the immunogeniccompositions, the present inventors have been able to reduce the extentof changes observed when the immunogenic compositions are stored.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All documents referred to herein, including patents and patentapplications, are incorporated by reference in their entirety.

1. An immunogenic composition comprising an Rv1196 related antigen,wherein: (i) the conductivity of the composition is 13 mS/cm or lower;and/or (ii) the concentration of salts in said composition is 130 mM orlower; and/or (iii) the concentration of sodium chloride in saidcomposition is 130 mM or lower.
 2. (canceled)
 3. The immunogeniccomposition according to claim 1, wherein the conductivity of thecomposition is 3 mS/cm or lower.
 4. (canceled)
 5. The immunogeniccomposition according to claim 1, wherein the concentration of salts insaid composition is 40 mM or lower.
 6. (canceled)
 7. The immunogeniccomposition according to claim 1, wherein the concentration of sodiumchloride in said composition is 40 mM or lower. 8-10. (canceled)
 11. Theimmunogenic composition according to claim 1, further comprising anon-ionic tonicity agent.
 12. The immunogenic composition according toclaim 11, wherein the non-ionic tonicity agent is a polyol. 13.(canceled)
 14. The immunogenic composition according to claim 12,wherein the polyol is sorbitol and wherein the concentration of sorbitolis between about 4 and about 6% (w/v).
 15. The immunogenic compositionaccording to claim 14, wherein the concentration of sucrose is betweenabout 4 and about 6% (w/v).
 16. The immunogenic composition according toclaim 1, further comprising one or more immunostimulants. 17-18.(canceled)
 19. The immunogenic composition according to claim 16,wherein the immunogenic composition comprises saponin is QS21.
 20. Theimmunogenic composition according to claim 16, wherein the immunogeniccomposition comprises 3-de-O-acylated monophoshoryl lipid A.
 21. Theimmunogenic composition according to claim 1, wherein the osmolality is250 to 750 mOsm/kg. 22-23. (canceled)
 24. The immunogenic compositionaccording to claim 1, wherein the composition is provided as a unit doseof between 50 ul and 1 ml and wherein the unit dose contains 1 to 100 ugof Rv1196 related protein.
 25. (canceled)
 26. The immunogeniccomposition according to claim 13, wherein the pH of said composition isin the range 7.0 to 9.0.
 27. (canceled)
 28. The immunogenic compositionaccording to claim 1, wherein the Rv1196 related antigen comprises asequence having at least 90% identity to SEQ ID No:
 1. 29. Theimmunogenic composition according to claim 1, wherein the Rv1196 relatedantigen comprises a fragment of SEQ ID No: 1 which is at least 350 aminoacids in length. 30-34. (canceled)
 35. A pharmaceutical compositioncomprising the immunogenic composition according to claim
 1. 36.(canceled)
 37. A method for the prophylaxis, treatment or ameliorationof infection by mycobacteria, such as infection by Mycobacteriumtuberculosis, comprising the administration of a safe and effectiveamount of an immunogenic composition according to claim
 1. 38. A processfor the making an immunogenic composition according to claim 1comprising the steps: a) lyophilising an Rv1196 related antigen; and b)reconstituting the lyophilised Rv1196 related antigen of step a) with anaqueous solution wherein: (i) the conductivity of the solution is 13mS/cm or lower; and/or (ii) the concentration of salts in said solutionis 130 mM or lower; and/or (iii) the concentration of sodium chloride insaid solution is 130 mM or lower.
 39. A method for the manufacture of astable immunogenic composition according to claim 1 comprising thesteps: a) lyophilising an Rv1196 related antigen; and b) reconstitutingthe lyophilised Rv1196 related antigen of step a) with an aqueoussolution wherein: (i) the conductivity of the solution is 13 mS/cm orlower; and/or (ii) the concentration of salts in said solution is 130 mMor lower; and/or (iii) the concentration of sodium chloride in saidsolution is 130 mM or lower; wherein at least 80% of the antigenicity ofthe immunogenic composition remains after storage at 25° C. for a periodof 24 hours.
 40. A kit comprising: a) a lyophilised Rv1196 relatedantigen; and b) an aqueous solution wherein: (i) the conductivity of thesolution is 13 mS/cm or lower; and/or (ii) the concentration of salts insaid solution is 130 mM or lower; and/or (iii) the concentration ofsodium chloride in said solution is 130 mM or lower. 41-46. (canceled)